Adiponectin peptidomimetics for treating ocular disorders

ABSTRACT

Provided herein are compositions and methods for treating dry eye or an ocular disease associated with inflammation in a subject in need thereof. The therapeutic compositions comprise an adiponectin peptidomimetic compound, and a pharmaceutically acceptable carrier, and administering a therapeutic agent. Also provided are methods for alleviating one or more symptoms or clinical signs of dry eye or an ocular disease associated with inflammation in a subject in need thereof.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to and is a Continuation In Part ofcurrently PCT Application No. PCT/US2016/030142, entitled, ADIPONECTINPEPTIDOMIMETICS FOR TREATING OCULAR DISORDERS, naming Henry Hsu asinventor, filed Apr. 29, 2016, which published as WIPO Publication No.WO 2016/179007 on Nov. 10, 2016 and claims priority to U.S. ProvisionalApplication No. 62/156,127. filed May 1, 2015, the disclosures of whichare hereby incorporated by reference in their entirety for all purposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

This application includes a Sequence Listing as a text file named“SEQLIST_098102-000110PC-1006637_ST25.txt” created Apr. 28, 2016, andcontaining 5,295 bytes. The material contained in this text file isincorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

The ocular surface system consists of the cornea, conjunctiva, lacrimalglands, meibomian glands, nasolacrimal duct, and their associated tearand connective tissue matrices, as well as the eyelids and eyelashes,all integrated by continuous epithelia and interconnected nervous,endocrine, immune, and vascular systems. Human tears are produced by thelacrimal glands. Tears are distributed by blinking, undergo evaporationfrom the ocular surface, and drain through the nasal lacrimal duct.Tears comprise three layers: an innermost layer of hydrophilic mucin, aslimy substance produced by the goblet cells that coats the ocularsurface epithelium; an aqueous tear layer produced by the lacrimalglands which floats on the mucin layer and is approximately 0.9% saline;and a superficial thin lipid layer produced by the meibomian glands,which helps with uniform tear spreading and to slow down tearevaporation. This three-layer structure stabilizes the tear film andenables the tear film to keep the eye moist, create a smooth surface forlight to pass through the eye, nourish the front of the eye, and provideprotection from injury and infection. Factors that disturb the delicatehomeostatic balance of the ocular surface system can adversely affecttear film stability and osmolarity, resulting in osmotic, mechanical,and inflammatory damage. Exposure of ocular surface epithelial cells toelevated tear osmolarity activates inflammatory pathways including therelease of pro-inflammatory cytokines. This can lead to the recruitmentand infiltration of immune cells to the ocular surface, particularlyantigen presenting cells and T cells.

Dry eye disease (DED or dry eye), also known as keratoconjunctivitissicca, is a multifactorial disorder of the tears and ocular surface. Itis characterized by symptoms including dry irritated eyes, excessivelywatery eyes, burning and stinging, light sensitivity, a foreign bodysensation, pain and redness, eye fatigue, and/or blurred vision. In dryeye the ocular surface epithelium undergoes squamous metaplasia,manifested by loss of goblet cells, mucin deficiency and keratinization,resulting in tear film instability. Factors that adversely affect tearfilm stability and osmolality can induce ocular surface damage andinitiate an inflammatory cascade that generates innate and adaptiveimmune responses. These immuno-inflammatory responses lead to furtherocular surface damage and the development of a self-perpetuatinginflammatory cycle (Stevenson et al., Arch Ophthalmol. 2012,130(i):90-100).

The major classes of dry eye are aqueous tear-deficient dry eye (ADDE)and evaporative dry eye (EDE). ADDE is due to failure of lacrimal tearsecretion and this class can be further subdivided to Sjogren's syndromedry eye (the lacrimal and salivary glands are targeted by an autoimmuneprocess, e.g., rheumatoid arthritis) and non-Sjogren's syndrome dry eye(lacrimal dysfunction, but the systemic autoimmune features of Sjogren'ssyndrome are excluded, e.g., age-related dry eye). EDE is due toexcessive water loss from the exposed ocular surface in the presence ofnormal lacrimal secretory function. Its causes can be intrinsic (due tointrinsic disease affecting lid structures or dynamics, e.g., meibomiangland dysfunction) or extrinsic (where ocular surface disease occurs dueto some extrinsic exposure, e.g., vitamin A deficiency). With meibomiangland dysfunction, the lipid layer of tears is altered, causingincreased tear evaporation. {See, e.g., “The Definition andClassification of Dry Eye Disease: Guidelines from the 2007International Dry Eye Work Shop,” Ocul Surf, 2007, 5(2): 75-92). In bothclasses of dry eye, the end result is a self-perpetuating cycle ofirritation and inflammation.

It is estimated that almost 5 million Americans 50 years and older haveDED, and millions more experience episodic symptoms of dry eye; ofthese, approximately two-thirds are women. The prevalence of DED risesdramatically with increasing age. Dry eye disease can hinder theperformance of activities of daily living, and DED is associated with anoverall decrease in quality of life.

There are several techniques or clinical measures used for diagnosingand evaluating the severity of a patient's dry eye, including the OcularSurface Disease Index (OSDI) questionnaire, the Symptom Assessment inDry Eye (SA DE), Tear Break-up Time (TBUT), vital dye staining of theocular surface, tear meniscus height analysis, tear film osmolarityanalysis, the Schirmer's Test, and the like. The TBUT test measures thetime required for the three-layer tear film to break up. A shortenedTBUT test time indicates a decreased quality of tears and is indicativeof dry eye. The Schirmer's Test measures the volume of tears produced,and is performed by of placing a small strip of filter paper inside thelower eyelid (conjunctival sac) of each eye for several minutes,allowing tear fluid to be drawn into the filter paper by capillaryaction. The paper is then removed and the amount of moisture is measuredin millimeters. Typically, a measurement of less than 5 mm indicates dryeye.

Ophthalmologists who treat chronic DED patients have to manage thesymptoms of ocular surface inflammation. Apart from reducing vision, thesymptoms of such inflammation also include redness, pain, swelling,edema (chemosis) of the conjunctiva and eyelids. In DED, the irritativesymptoms may be due to the release of pro-inflammatory cytokines (Lam etal, Am J Ophthalmol, 2009, 147: 198-205; Albersmeyer et al., Exp EyeRes, 2010, 90(3):444-451) and infiltration of inflammatory cells (Kunertet al, Arch. Ophthalmol. 2000, 118-(11): 1489-96) on the ocular surface,as well as stimulation of the nerve fibers innervating the ocularsurface, resulting ocular surface tissue damage. Inflammation also leadsto epitheliopathy, the key clinical sign identified in DED.

Current therapies for dry eye are palliative with a focus on thereplacement of tears to reduce symptoms. Conventional treatment of mildand moderate cases of dry eye includes supplemental lubrication.Application of ophthalmic formulations, such as therapeutic eye dropsand artificial tears, every few hours can aid in maintaining andstrengthening the tear film on the ocular surface and provide temporaryrelief. Lubricating tear ointments are also used. Tear ointments containwhite petrolatum, mineral oil, and similar lubricants, and serve as alubricant and an emollient. While these palliative therapies havebenefits over the short term, they have limited utility in long-termcontrol therapy for dry eye.

RESTASIS® (cyclosporine A) is the first prescription product for dry eyetherapy. Cyclosporine A exerts immunosuppressive activity throughseveral pathways and the immunomodulatory activity of cyclosporine A isused in the treatment of immune-based disorders, such as transplantrejection, psoriasis, ulcerative colitis, rheumatoid arthritis, and DED.Topical administration of cyclosporine A has been shown to increase tearfluid secretion, possibly by promoting the local release ofparasympathetic nervous system-associated neurotransmitters. Thebeneficial effects of cyclosporine A treatment in DED are wellestablished; however, it is clear that many patients with DED do notshow a consistent therapeutic response to topical cyclosporine A.

Thus, there are currently few effective therapeutic options for themajority of patients with dry eye and ocular diseases associated withinflammation. As such, there is a high unmet need for effective and safetherapies. The present invention satisfies this need and provides otheradvantages as well.

BRIEF SUMMARY OF THE INVENTION

In one aspect, provided herein are compositions and methods for treatingdry eye in a subject in need thereof. The method comprises administeringto the subject a therapeutically effective amount of compositioncomprising: an adiponectin peptidomimetic compound and a Lifitegrast anda pharmaceutically acceptable carrier to treat dry eye in the subject.

In some embodiments, the composition and therapeutic agent areadministered topically, by intravitreal injection, by subconjunctivalinjection, by conjunctival injection, by intramuscular injection, bysubcutaneous injection, by intravenous injection, by intracameralinjection, or by implantation into the subject's eye. In someembodiments, the dry eye is selected from the group consisting ofhypolacrimation, tear deficiency, xerophthalmia, Sjogren's syndrome dryeye, non-Sjogren's syndrome dry eye, keratoconjuctivitis sicca, aqueoustear-deficiency dry eye (ADDE), evaporative dry eye (EDE), environmentaldry eye, Stevens-Johnson syndrome, ocular pemphigoid, blepharitismarginal, eyelid-closure failure, sensory nerve paralysis, allergicconjunctivitis-associated dry eye, post-viral conjunctivitis dry eye,post-cataract surgery dry eye, VDT operation-associated dry eye, andcontact lens wearing-associated dry eye.

In some instances, the composition and therapeutic agent are eachadministered to the subject once a day, two times a day, three times aday, four times a day, or more often (more frequently). In otherinstances, the composition and therapeutic agent are each administeredevery other day or less often (less frequently). In some embodiments,the adiponectin peptidomimetic compound is present in an amount betweenabout 0.0001% (wt) to about 90% (wt) of the final composition. In someembodiments, the therapeutic agent is present in an amount between about0.1% to about 20%. In some embodiments, the composition and therapeuticagent are each in a formulation selected from the group consisting of asolution, suspension, syrup, liquid, gel, hydrogel, emulsion, liposome,aerosol, mist, film, suspension, plug, polymer, implant, contact lens,ocular insert, nanoparticle, microparticle, a sustained releaseformulation, and a formulation suitable for an ocular medical device. Insome embodiments the method further comprises administering acomposition comprising cyclosporine, artificial tears, a corticosteroid,an anti-inflammatory agent, or any combination thereof.

In some embodiments, the adiponectin peptidomimetic compound isrepresented by Formula II:Xaai-Ile-Pro-Xaa₂-Leu-Tyr-Xaa₃-Phe-Ala-Xaa₄-Xaa₅ (SEQ ID NO:2) (II);wherein the C-terminal amino acid is optionally amidated; a variantthereof; a derivative thereof; or a pharmaceutically acceptable saltthereof. In some instances, the adiponectin peptidomimetic compound isselected from the group consisting ofD-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser (SEQ ID NO:3),D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser-p-Ala (SEQ ID NO:4),D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser-p-Ala-NH₂ (SEQ ID NO:5),D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser-NH₂ (SEQ ID NO:6),(D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser-His-Pro)₂-Dab-NH₂ (SEQ IDNO:7), a variant thereof, a derivative thereof, and a pharmaceuticallyacceptable salt thereof, wherein Dab represents 2,3-diamino butyricacid). In some embodiments, the adiponectin peptidomimetic compound isADP 355 (SEQ ID NO:6), or a pharmaceutically acceptable salt thereof.

In one embodiment, the therapeutic agent is selected from Lifitegrastand Restasis® or a pharmaceutically acceptable salt thereof. In someembodiments, the therapeutic agent comprises about 3.5% to about 6.5%Lifitegrast.

In another aspect, provided herein is a method for treating an oculardisease associated with inflammation in a subject in need thereof. Themethod comprises administering to the subject a therapeuticallyeffective amount of a composition comprising an adiponectinpeptidomimetic compound (SEQ ID NO:6) or a pharmaceutically acceptablesalt thereof, and a pharmaceutically acceptable carrier, andadministering a therapeutic agent to treat the ocular disease associatedwith inflammation in the subject.

In another aspect, provided herein is a method for alleviating at leastone symptom or clinical sign of dry eye in a subject in need thereof.The method comprises administering to the subject a therapeuticallyeffective amount of an adiponectin peptidomimetic compound (SEQ ID NO:6)or a pharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier; and administering a therapeutic agent to alleviateat least one symptom or clinical sign of dry eye in the subject.

In yet another aspect, provided herein is a method for treating anocular disease or disorder in a subject in need thereof. The methodcomprises administering to the subject a therapeutically effectiveamount of an adiponectin peptidomimetic compound or a pharmaceuticallyacceptable salt thereof: and a pharmaceutically acceptable carrier; andadministering a therapeutic agent to treat the ocular disease associatedwith inflammation in the subject.

In another aspect, the subject can present with at least one symptom orclinical sign of dry eye selected from the group consisting of a changein tear secretion, a change in tear clearance, ocular surface damage,corneal epithelial defects, a change in ocular surface cells, a changein tear film stability, a change in tear volume, a change in tear filmcomposition, a change in tear osmolarity, and any combination thereof.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that administration of an adiponectin peptidomimeticto a mouse model of experimental dry eye (EDE) increased tear volumecompared to control untreated EDE mice. Tear volumes were measured atday 5 and day 10 after treatment initiation. Tear volumes in theadiponectin peptidomimetic-treated animals were similar to that ofuntreated normal mice. Also, tear volume was significantly improved inthe treated EDE mice compared to control untreated EDE mice. “UT”represents untreated (normal) mice; “EDE” represents untreatedexperimental dry eye control mice; “BSS only” represents experimentaldry eye mice treated with only balanced salt solution; “ADP355”represents experimental dry eye mice treated with adiponectinpeptidomimetic; “ADP399” represents experimental dry eye mice treatedwith adiponectin peptidomimetic (linear branched dimer); and “gAdipo”represents experimental dry eye mice treated with recombinant murinefull-length globular adiponectin.

FIG. 2 shows that adiponectin peptidomimetic treatment minimized ordecreased corneal surface irregularity in a mouse model of experimentaldry eye, compared to control untreated EDE mice. Fluorescein stainingwas used to evaluate the smoothness of the corneal surface. The cornealsurface was assessed at day 5 and day 10 after EDE initiation. “UT”represents untreated (normal) eye mice; “EDE” represents untreatedexperimental dry eye control mice; “BSS only” represents experimentaldry eye mice treated with only balanced salt solution; “ADP355”represents experimental dry eye mice treated with adiponectinpeptidomimetic; “ADP399” represents experimental dry eye mice treatedwith adiponectin peptidomimetic (linear branched dimer); and “gAdipo”represents experimental dry eye mice treated with recombinant murinefull-length globular adiponectin.

FIGS. 3A and 3B depict bar charts illustrating the change in tear filmbreak-up time when dry eye is untreated, normal (UT) and treated with0.1% ADP 355, 5% Lifitegrast, and both 0.1% ADP 355 +5% Lifitegrast.FIG. 3A after treating for 5 days. FIG. 3B, after 10 days treatment.

FIGS. 4A and 4B depict bar charts illustrating changes in cornealstaining when dry eye is untreated, normal (UT) and treated with either0.1% ADP 355, 5% Lifitegrast, and both 0.1% ADP 355+Lifitegrast 5%. FIG.4A, after treating for 5 days. FIG. 3B, after 10 days treatment.

FIG. 5B depicts scatter plots illustrating the change in Activated Tcell levels when dry eye presents as conjunctiva. FIG. 5A representsscatter plots of activated T cell level of normal (UT) eye. Aftertreatment, the changes in activated T cell levels are illustrated inscatter plots of FIG. 5C (after administration of 0.1% ADP 355), FIG. 5D(after administration of 5% Lifitegrast), and FIG. 5E (afteradministration of both 0.1% ADP 355+Lifitegrast 5%).

FIG. 6B depicts scatter plots illustrating the change in activated Tcell levels when dry eye presents in the lacrimal gland. FIG. 6Arepresents scatter plots of activated T cell level of normal (UT) eye.After treatment, the changes in activated T cell levels are illustratedin scatter plots of FIG. 6C (after administration of 0.1% ADP 355), FIG.6D (after administration of 5% Lifitegrast), and FIG. 6E (afteradministration of both 0.1% ADP 355+Lifitegrast 5%).

FIG. 7 is table of cytokine levels identified in both the conjunctivaand lacrimal gland of normal (UT) and dry eyes with and withouttherapeutic intervention.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

Provided herein are compositions, methods and kits for treating dry eyeor an ocular disease associated with inflammation in a subject in needthereof. The method includes administering to said subject atherapeutically effective composition comprising an adiponectinpeptidomimetic compound or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier and administering atherapeutic agent. Also provided herein are methods for alleviating atleast one symptom or clinical sign of dry eye in a subject. In someembodiments, the therapeutically effective composition comprises atleast two different adiponectin peptidomimetic compounds orpharmaceutically acceptable salts thereof and administering atherapeutic agent. The invention is based, in part, on the discoverythat administration to the eye of an adiponectin peptidomimetic compoundor a pharmaceutically acceptable salt thereof and administering atherapeutic agent increases tear volume and reduces corneal surfaceirregularities in subjects with dry eye.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The terms “a,” “an,” or “the” as used herein not only include aspectswith one member, but also include aspects with more than one member. Forinstance, the singular forms “a,” “an,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the agent” includes reference to one or more agents knownto those skilled in the art, and so forth.

The term “dry eye” refers to a multifactorial disease of the tears andocular surface (including the cornea, conjunctiva, and eye lids) resultsin symptoms of discomfort, visual disturbance and tear film instabilitywith potential damage to the ocular surface, as defined by the “TheDefinition and Classification of Dry Eye Disease: Guidelines from the2007 International Dry Eye Work Shop,” Ocul Surf, 2007, 5(2): 75-92).Dry eye can be accompanied by increased osmolarity of the tear film andinflammation of the ocular surface. Dry eye includes dry eye syndrome,keratoconjunctivitis sicca (KCS), dysfunctional tear syndrome, lacrimalkeratoconjunctivitis, evaporative tear deficiency, aqueous teardeficiency, and LASIK-induced neurotrophic epitheliopathy (LE).

The term “ocular disease associated with inflammation” refers to adisease or disorder of the eye wherein inflammation causes damage to theocular surface system. As used herein, “the ocular surface system”includes the cornea, conjunctiva, lacrimal glands, meibomian glands,nasolacrimal duct, and their associated tear and connective tissuematrices, as well as the eyelids and eyelashes, all integrated bycontinuous epithelia and interconnected nervous, endocrine, immune, andvascular systems.

The term “a symptom” refers to a subjective indication or observation ofa disorder or disease experienced or perceived by a patient.

The term “a clinical sign” refers to an objective indication,observation or evidence of a disorder or a disease that may be detectedor interpreted by a clinician.

The term “adiponectin” refers to a polypeptide that is primarily derivedfrom adipocytes. The adiponectin polypeptide is composed of 244 aminoacid residues containing a short non-collagenous N-terminal segment(about 130 amino acids) followed by a collagenI like sequence (Maeda etal., BBRC, 1996, 221 :286-289). The amino acid sequence of humanadiponectin polypeptide is found, for example, in NCBI Ref. Sequence No.NP 004788.1 or UniPro Ref. No. Q15848. Adiponect n can form a homotrimerthat is similar in size and overall structure to complement protein Clq,with particularly high homology (about 65-70% homology) to Clq in theC-terminal globular domain. This globular domain (about 130 amino acids)is believed to be essential for the biological activity of natural(native) adiponectin. The crystal structure of adiponectin revealedadditional high structural similarity between this same globular domainand TNFa (about 60% homology).

The term “an adiponectin peptidomimetic” refers to a peptide compoundthat mimics the activity or function of adiponectin protein. Anadiponectin peptidomimetic may have the ability to bind to or interactwith one or more adiponectin receptors (AdipoRI and AdipoR2) or variantsthereof. A peptidomimetic may be a backbone modified peptide, anypolyamide or other polymeric structure resembling peptides, peptidescontaining non-natural amino acid residues or a peptide derivative.

The term “peptide” refers to an organic compound comprising a chain oftwo or more amino acids covalently joined by peptide bonds. Peptides maybe referred to with respect to the number of constituent amino acids,i.e., a dipeptide contains two amino acid residues, a tripeptidecontains three, etc.

The term “amino acid” as used herein means an organic compoundcontaining both a basic amino group and an acidic carboxyl group.Included within this term are natural amino acids {e.g., L-amino acids),modified and unusual amino acids {e.g., D-amino acids), as well as aminoacids which are known to occur biologically in free or combined form butusually do not occur in proteins. Included within this term are modifiedand unusual amino acids, such as those disclosed in, for example,Roberts and Vellaccio (1983) The Peptides, 5: 342-429, the teaching ofwhich is hereby incorporated by reference. Natural protein occurringamino acids include, but are not limited to, alanine, arginine,asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine,histidine, isoleucine, leucine, lysine, methionine, phenylalanine,serine, threonine, tyrosine, tyrosine, tryptophan, proline, and valine.Natural non-protein amino acids include, but are not limited toarginosuccinic acid, citrulline, cysteine sulfinic acid,3,4-dihydroxyphenylalanine, homocysteine, homoserine, ornithine,3-monoiodotyrosine, 3,5-diiodotryosine, 3,5,5′-triiodothyronine, and3,3′,5,5′-tetraiodothyronine. Modified or unusual amino acids which canbe used to practice the invention include, but are not limited to,D-amino acids, hydroxylysine, 4-hydroxyproline, an N-Cbz-protected aminoacid, 2,4-diaminobutyric acid, homoarginine, N-methyl-arginine,norleucine, N-methylaminobutyric acid, naphthylalanine, phenylglycine,beta-phenylproine, tert-leucine, 4-aminocyclohexylalanine,N-methyl-norleucine, norvaline, 3,4-dehydroproline,N,N-dimethylaminoglycine, N-methylaminoglycine,4-aminopiperidine-4-carboxylic acid, 6-aminocaproic acid,trans-4-(aminomethyl)-cyclohexanecarboxylic acid, 2-, 3-, and4-(aminomethyl)-benzoic acid, 1-aminocyclopentanecarboxylic acid,1-aminocyclopropanecarboxylic acid, and 2-benzyl-5-aminopentanoic acid.

The term “hydrophobic residue” includes valine, isoleucine, leucine,methionine, phenylalanine, tyrosine, tryptophan, and functionalequivalents thereof.

The term “polar residue” includes aspartic acid, asparagine, glutamicacid, glutamine, lysine, arginine, histidine, serine, and functionalequivalents thereof.

The term “peptide bond” means a covalent amide linkage formed by loss ofa molecule of water between the carboxyl group of one amino acid and theamino group of a second amino acid.

The term “peptide backbone” means the chain of atoms of a peptidecomprising the carboxamide groups that are the peptide bonds togetherwith the atoms of the amino acids that link the carboxyl and aminogroups of the amino acid (usually the a-carbon of an a-amino acid).

The term “side chain” means groups that are attached to the peptidebackbone, and typically refers to the group attached to the a-carbon ofan .alpha. -amino acid. For example, for the side chains of theproteinogenic amino acids include: methyl (alanine), hydroxymethyl(swine), benzyl (phenylalanine), mercaptomethyl (cysteine), andcarboxymethyl (aspartic acid).

The term “derivative” as applied to compounds comprising a peptide chainmeans a compound wherein one or more of the amino, hydroxyl, or carboxylgroups in a side chain of the peptide, or the terminal amino or carboxylgroups, is modified to a derivative functional group. An amino group maybe derivatized as an amide (such as an alkyl carboxamide, acetamide), acarbamate (such as an alkyl carbamate, e.g. methyl carbamate ort-butylcarbamate), or a urea. A hydroxyl group may be derivatized as anester (such as an alkanoate, e.g. acetate, propionate, or anarenecarboxylate, e.g. benzoate), a carbamate (such as an alkylcarbamate, e.g. methyl carbamate), a carbonate (such as an alkylcarbonate, e.g. ethyl carbonate. A carboxyl group may be derivatized asan ester (such as an alkyl ester, e.g. ethyl ester) or an amide (e.g.primary carboxamide, an N-alkyl secondary carboxamide, or anN,N-dialkylcarboxamide). A person skilled in the art will appreciatethat derivatives of the peptide will be expected to result in retentionof the properties of the parent peptide, either because theincorporation of the derivative group does not change the properties ofthe peptide, or the derivatizing group is removed in vivo (e.g. viametabolism). Preferred embodiments of the invention are those whereinthree or fewer of the amino, carboxyl, and hydroxyl groups, andpreferably two or fewer, or one or none, are modified to a derivativefunctional group. The term “derivative” also includes salts ofderivatives.

The term “non-natural amino acid” is used to refer to an amino acidwhich does not exist on its own in nature, but rather, has beensynthesized or created by man. Examples of non-natural amino acidsinclude iodinated tyrosine, methylated tyrosine, glycosylated serine,glycosylated threonine, azetidine-2-carboxylic acid, 3,4-dehydroproline,perthiaproline, canavanine, ethionine, norleucine, selenomethionine,animohexanoic acid, telluromethionine, homoallylglycine, andhomopropargylglycine. D-amino acids are also examples of non-naturalamino acids.

“Nva” corresponds to the non-natural amino acid norvaline, also known as2(L)-aminopentanoic acid. “NvaNH₂” corresponds to 2(L)-aminopentanamide.“Acp” corresponds to the non-natural amino acid 6-aminocaproic acid,also known as 6-amino-hexanoic acid. “Acp H₂” corresponds to6-aminocapramide, also known as 6-amino-hexanamide. “Dpr(Ac)”corresponds to N2(3)-acetyl-diaminopropionic acid. “Dbu” corresponds to2,4-diaminobutyric acid. “Glc” corresponds to glucose. “PGlc”corresponds to beta-glucose. “SerP(Glc)” corresponds to serineglycosylated with a beta-glucosyl residue on the alcohol hydroxyl group.“Thr(NAcGal)” corresponds to threonine glycosylated with an N-acetylgalactosaminyl residue on the alcohol hydroxyl group. “Tyr(I₂)”corresponds to 3,5-diiodotyrosine. “N-MeArg” corresponds toN-methyl-arginine. “PAla” corresponds to beta-alanine, also known as3-aminopropanoic acid. “PAla-H₂” corresponds to the amide derivative ofbeta-alanine, also known as 3-aminopropanamide. “(D)-Ser” corresponds toD-serine. “Apa” corresponds to aminopentanoic acid. “AlloThr”corresponds to allo-threonine, also known as(2S,3S)-2-amino-3-hydroxybutanoic acid. “3Hyp” corresponds to3-hydroxyproline. “4Hyp” corresponds to 4-hydroxyproline.

As used herein, the term “hydroxylated acyclic amino acid” refers to anacyclic amino acid that contains at least one alcohol hydroxyl group inits structure. Preferred, but non-limiting, examples of hydroxylatedacyclic amino acid are serine, (D)-serine, threonine, (D)-threonine,(L)-allo-threonine, (D)-allo-threonine, (L)-isoserine, (D)-isoserine,(i)-β-homoserine, (D)-P-homoserine, (L)-homoserine, and (D)-homoserine.

As used herein, the term “aliphatic amino acid” refers to an amino acidwhich carbon chain is aliphatic in nature. Non-limiting examples ofaliphatic amino acids are alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine,lysine, methionine, proline, serine, threonine, valine, Nva, Nva-NH₂,Acp, Acp-NH₂, Dpr(Ac), Abu, N-MeArg, pAla, pAla-NH₂, Apa, and AlloThr.Preferred aliphatic amino acids within the present application are pAla,pAla-NH₂, Acp and Acp-NH₂.

The term “peptide transduction domain” is used to indicate a peptide, orderivative thereof, that is capable of crossing cell membranes and ofdirecting the transport of a peptide, protein, or molecule associatedwith the protein transduction domain, from the outside of a cell intothe cytoplasm of the cell through the cytoplasmic membrane of the cell.

The term “conjugated” referring to the linking of two peptides meansthat the two peptides are covalently linked to one another. The linkingmay be accomplished directly, through the formation of an amide bondbetween the carboxyl group of one peptide and an amino group of theother peptide, or by means of a linking group wherein the linking grouphas covalent bonds to each of the peptides. For example, the linkinggroup may be a peptide chain, an amino acid, or any group having atleast two functional groups and capable of forming covalent bond to eachof the two peptide chains.

An “acetylated amino acid” as used herein refers to an amino acid havingan acetyl moiety in its side chain.

As used herein, “pharmaceutically-acceptable” refers to those compounds,materials, compositions, and/or dosage forms that are, within the scopeof sound medical judgment, suitable for contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem complications commensurate with areasonable benefit/risk ratio.

The term “pharmaceutically acceptable salt” refers to a derivative ofthe disclosed compounds wherein the parent compound is modified bymaking acid or base salts thereof, including acid addition salts andbase addition salts. Examples of pharmaceutically-acceptable saltsinclude, but are not limited to, mineral or organic acid salts of basicresidues such as amines; alkali or organic salts of acidic residues suchas carboxylic acids; and the like. The term “acid addition salt” refersto the corresponding salt derivative of a parent compound that has beenprepared by the addition of an acid. The pharmaceutically-acceptablesalts include the conventional salts or the quaternary ammonium salts ofthe parent compound formed, for example, from inorganic or organicacids. For example, such conventional salts include, but are not limitedto, those derived from inorganic acids such as hydrochloric,hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; andthe salts prepared from organic acids such as acetic, propionic,succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic,pamoic, maleic, adipic, alginic, aspartic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,benzenesulfonic, toluenesulfonic, methanesulfonic, 2-napthalenesulfonic,ethane disulfonic, oxalic, isethionic, glucoheptanoic,glycerophosphoric, hemisulfanic, heptanoic, hexanoic, hydrochloric,hydrobromic, hydroiodic, 2-hydroxyethanesulfonic, 2-napthalenesulfonic,pectinic, phosphoric, sulfuric, 3-phenylpropionic, picric, pivalic,thiocyanic, p-toluenesulfonic, butyric, camphoric, camphorsulfonic,digluconic, cyclopentanepropionic, bisulfuric, dodecylsulfuric,ethanesulfonic, and undecanoic and the like. Thus, the term “baseaddition salt” refers to the corresponding salt derivative of a parentcompound that has been prepared by the addition of a base. Also, thebasic nitrogen-containing groups can be quaternized with such agents aslower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride,bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl,and diamyl sulfates, long chain halides such as decyl, lauryl, myristyland stearyl chlorides, bromides and iodides, aralkyl halides like benzyland phenethyl bromides, and others. The pharmaceutically-acceptablesalts include the conventional salts or the quaternary ammonium salts ofthe parent compound formed, for example, from inorganic or organicbases. For example, such conventional salts include, but are not limitedto, those derived from inorganic bases such as lithium hydroxide, sodiumhydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxideand ammonium hydroxide and the salts prepared from organic amines, suchas methyl amine, ethyl amine, isopropyl amine, piperidine, piperizine,pyrrolidine, ethanolamine, morpholine, diazapine, ethylene diamine,pyridine, quinoline, quinuclidine, and the like.

The term “therapeutically effective amount,” “effective amount” or“therapeutically effective dose” refers to that amount of thetherapeutic agent sufficient to ameliorate a disorder, as describedabove. For example, for the given parameter, a therapeutically effectiveamount will show an increase or decrease of therapeutic effect at least5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.Therapeutic efficacy can also be expressed as “-fold” increase ordecrease. For example, a therapeutically effective amount can have atleast a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over acontrol. In the context of the present invention, the effective amountof an adiponectin peptidomimetic compound can vary depending onco-administration of other therapeutics or disease profile of theindividual (among other factors such as age, severity of disease, etc.).

The terms “therapy,” “treatment,” and “amelioration” refer to anyreduction in the severity of symptoms. In the case of treating an oculardisorder, e.g., dry eye, the terms can refer to adding artificial tears,conserving tears, reducing tear evaporation, increasing tear production,reducing inflammation of the eyelids or eye surface, reducing ocularsigns to dry eye, etc. As used herein, the terms “treat” and “prevent”are not intended to be absolute terms. Treatment can refer to any delayin onset, amelioration of symptoms, improvement in patient's quality oflife, etc. The effect of treatment can be compared to an individual orpool of individuals not receiving the treatment, or to the same patientprior to treatment or at a different time during treatment. In someaspects, the severity of disease is reduced by at least 10%), ascompared, e.g., to the individual before administration or to a controlindividual not undergoing treatment. In some aspects the severity ofdisease is reduced by at least 25%, 50%, 75%), 80%), or 90%, or in somecases, no longer detectable using standard diagnostic techniques.

The term “treating” or “treatment” refers to the treating or treatmentof a disease or medical condition (such as dry eye or an ocular diseaseassociated with inflammation) in a patient, such as a mammal(particularly a human or an animal) which includes: ameliorating thedisease or medical condition, i.e., eliminating or causing regression ofthe disease or medical condition in a patient; suppressing the diseaseor medical condition, i.e., slowing or arresting the development of thedisease or medical condition in a patient; or alleviating one or moresymptoms of the disease or medical condition in a patient. The termencompasses the prophylactic treatment of a disease or condition as toprevent or reduce the risk of acquiring or developing a specific diseaseor condition, or to prevent or reduce the risk of recurrence.

As used herein, “dosage unit” refers to physically discrete units suitedas unitary dosages for the particular patient to be treated. Each unitmay contain a predetermined quantity of active compound(s) calculated toproduce the desired therapeutic effect(s) in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention may be dictated by (a) the unique characteristicsof the active compound(s) and the particular therapeutic effect(s) to beachieved, and (b) the limitations inherent in the art of compoundingsuch active compound(s).

The term “subject,” “individual” or “patient” typically includes humans,but can also include other animals such as, e.g., other primates,rodents, canines, felines, equines, ovines, porcines, and the like.

III. Detailed Description of Embodiments

A. Adiponectin Peptidomimetic Compounds

Adiponectin is a 244 amino acid long polypeptide protein secreted mainlyby the adipose tissue. This relatively large 30 kDa protein isstructurally similar to tumor necrosis factor alpha (TNFα).

Adiponectin protein based biological modulators are still not available,partly due to difficulties in converting the full size adiponectinprotein into a viable systemic regulator. The main reason of thenon-drug ability of adiponectin protein is the extreme insolubility ofthe C-terminal domain and larger peptide fragments thereof. In addition,its relatively large size, and its propensity to form higher ordercomplexes that alter its receptor binding affinities, poses significantchallenges towards pharmaceutical manufacturing.

In one aspect of the invention, provided herein is an adiponectinpeptidomimetic compound. In some embodiments, the compound is a compoundrepresented by Formula I: X-M₁-SEQ ID NO: I-M₂-Z (I); wherein SEQ ID NO:1 is Xaai-Ile-Pro-Xaa₂-Leu-Tyr-Xaa₃-Phe-Ala-Xaa₄-Xaa₅. In someembodiments, Xaai is Asn or a non-natural amino acid; Xaa₂ is Gly or anon-natural amino acid; Xaa₃ is Tyr or a non-natural amino acid; Xaa₄ isTyr or a non-natural amino acid; and Xaa₅ is no amino acid, β-Ala orP-Ala-NH₂. In some instances, at least one of Xaai, Xaa₂, Xaa₃ or Xaa₄is a non-natural amino acid. For instance, Xaai, Xaa₂, Xaa₃ or Xaa₄ is anon-natural amino acid. In some embodiments, Xaai and Xaa₂, Xaai andXaa₃, Xaai and Xaa₄, Xaa₂ and Xaa₃, Xaa₂ and Xaa₄, and Xaa₃ and Xaa₄ arethe same or different non-natural amino acids. In other embodiments,Xaai and Xaa₂ and Xaa₃, Xaai and Xaa₂ and Xaa₄, Xaai and Xaa₃ and Xaa₄,and Xaa₂ and Xaa₃ and Xaa₄ are the same or different non-natural aminoacids. In yet other embodiments, Xaai and Xaa₂ and Xaa₃ and Xaa₄ are thesame or different non-natural amino acids. In some embodiments, X is anoptionally present 1-10 amino acid peptide (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 amino acid peptide), polymer molecule, lipophilic compound orpeptide transduction domain. In some embodiments, Z is an optionallypresent 1-10 amino acid peptide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10amino acid peptide), polymer molecule, lipophilic compound or peptidetransduction domain. In some embodiments, M₁ is an optionally presentsingle bond or a linking group. In some embodiments, M₂ is an optionallypresent single bond or a linking group. In some instances, the compoundof Formula I comprising a C-terminal amino acid, and the C-terminalamino acid can be optionally amidated. In some embodiments, any of thecompounds represented by Formula I are pharmaceutically acceptable saltsthereof.

In some embodiments, Xaai is D-Asn and Xaa₄ is D-Ser. In someembodiments, Xaa₂ is Nva. In some embodiments, Xaa₃ is D-Ser. In someembodiments, Xaa₂ is Nva and Xaa₃ is D-Ser. In some embodiments, Xaai isD-Asn; Xaa₂ is Nva; Xaa₃ is D-Ser and Xaa₄ is D-Ser.

In some embodiments, X and/or Z is a 1-10 amino acid peptide, e.g., 1amino acid peptide, 2 amino acid peptide, 3 amino acid peptide, 4 aminoacid peptide, 5 amino acid peptide, 6 amino acid peptide, 7 amino acidpeptide, 8 amino acid peptide, 9 amino acid peptide, or 10 amino acidpeptide. In some instances, the length of the peptides and identity ofthe substituent amino acids comprising the X and Z peptides, areindependently selected.

In some embodiments, Xaa₅ is β-Ala or P-Ala-NH₂. In some aspects, thecompound of Formula I is selected from the group consisting of ADP355,ADP355-PAIa, ADP355-pAla-NH₂, ADP355-NH₂, ADP399, variants thereof,derivatives thereof; and pharmaceutically acceptable salts thereof.Detailed descriptions of ADP355, ADP399, and other useful adiponectinpeptidomimetics are found, for example, in U.S. Pat. No. 9,073,965 andOtvos et al, Frontiers in Chemistry, 2014, 2(93): 1-15, doi:10.3386/fchem.2014.00093, the disclosure is hereby incorporated byreference in its entirety for all purposes.

In some embodiments, X or Z is a polymer molecule, a lipophilic compoundor an peptide transduction domain. In some embodiments, the polymer is alinear or branched polyethylene glycol. In other embodiments, thepolymer has a molecular weight of from 1 kDa to 200 kDa, such as 1 kDato 200 kDa, 50 kDa to 200 kDa, 100 kDa to 200 kDa, 1 kDa to 100 kDa, 1kDa to 50 kDa, 50 kDa to 100 kDa, 1 kDa, 50 kDa. 100 kDa, 150 kDa or 200kDa. In yet other embodiments, the polymer has a molecular weight offrom 2 kDa to 95 kDa, such as 2 kDa to 95 kDa, 10 kDa to 95 kDa, 20 kDato 95 kDa, 30 kDa to 95 kDa, 40 kDa to 95 kDa, 50 kDa to 95 kDa, 60 kDato 95 kDa, 70 kDa to 95 kDa, 80 kDa to 95 kDa, 2 kDa to 90 kDa, 2 kDa to80 kDa, 2 kDa to 70 kDa, 2 kDa to 60 kDa, 2 kDa to 50 kDa, 2 kDa to 40kDa, 2 kDa to 30 kDa, 2 kDa to 20 kDa, 2 kDa to 15 kDa, 2 kDa to 10 kDa,2 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, 80 kDa, 85 kDa, or90 kDa. In some embodiments, the polymer has a molecular weight of from5 kDa to 80 kDa, such as 5 kDa to 80 kDa, 5 kDa to 70 kDa, 5 kDa to 60kDa, 5 kDa to 50 kDa, 5 kDa to 40 kDa, 5 kDa to 30 kDa, 5 kDa to 20 kDa,5 kDa to 10 kDa, 70 kDa to 80 kDa, 60 kDa to 80 kDa, 50 kDa to 80 kDa,40 kDa to 80 kDa, 30 kDa to 80 kDa, 20 kDa to 80 kDa, or 10 kDa to 80kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 30 kDa, 35 kDa, 40 kDa, 45kDa, 50 kDa, 55 kDa, 60 kDa, 65 kDa, 70 kDa, 75 kDa, or 80 kDa. In otherembodiments, the polymer has a molecular weight of from 12 kDa to 60kDa, such as 12 kDa to 40 kDa, 20 kDa to 40 kDa, 12 kDa, 20 kDa, 30 kDa,40 kDa, 50 kDa or 60 kDa. The X and Z polymer molecules areindependently selected and may be the same or different.

In some embodiments, the polymer molecule is methoxyl PEG maleimide(mPEG(MAL)), methoxyl PEG forked maleimide (mPEG₂(MAL)), methoxyl PEGortho-pyridyldisulfide (mPEG-OPSS), PEG-vinyl sulphone, orortho-pyridyldisulfide-PEG-hydrazide (OPSS-PEG-hydrazide) in combinationwith methoxyl PEG aldehyde (mPEG-ALD). In other embodiments, the polymermolecule is selected from the group consisting of 5k-mPEG(MAL),20k-mPEG(MAL), 40k-mPEG₂(MAL), 5k-mPEG-OPSS, IOk-mPEG-mPSS,20k-mPEG-OPSS, or OPSS-PEG₂k-hydrazide in combination with mPEG₃₀kD-ALD.

One of skill in the art will appreciate that when X is not present, M₁will not be present either. One of skill in the art will appreciate thatwhen Z is not present, M₂ will not be present either. One of skill inthe art will appreciate that if the compound of Formula I has aC-terminus which comprises an amino acid, for example wherein theC-terminus comprises Z (if present and comprises a peptide or atransduction domain), Xaa₅ (if present) or Xaa₄, that amino acid isoptionally amidated. One of skill in the art will appreciate that apeptide (for example a dipeptide having two amino acids Xaai and Xaa₂)can be represented as: H-Xaai-Xaa₂-OH, wherein H is part of the freeamino terminus of the peptide and OH is part of the free carboxylterminus of the peptide; or the peptide can be represented as:Xaai-Xaa₂, wherein the H is part of the free amino terminus of thepeptide and the OH that is part of the free carboxyl terminus of thepeptide are not shown in the formula for the peptide, but are understoodto be present.

In other embodiments, the adiponectin peptidomimetic compound is acompound represented by Formula II:Xaai-Ile-Pro-Xaa2-Leu-Tyr-Xaa3-Phe-Ala-Xaa4-Xaa5 (SEQ ID NO:2) (II);wherein the C-terminal amino acid is optionally amidated; or apharmaceutically acceptable salt thereof. In some embodiments, theadiponectin peptidomimetic compound selected from the group consistingof D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser (SEQ ID NO:3),D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser-p-Ala (SEQ ID NO:4),D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser-p-Ala-H₂ (SEQ ID NO:5),D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-AIa-D-Ser-NH₂ (SEQ ID NO:6),(D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser-His-Pro)₂-Dab-NH₂ (SEQ IDNO:7, wherein Dab represents 2,3-diamino butyric acid), a variantthereof, a derivative thereof, and a pharmaceutically acceptable saltthereof.

Useful adiponectin peptidomimetic compounds are described in U.S. Pat.No. 9,073,965, and Otvos et al., Frontiers in Chemistry, 2014, 2(93):1-15, doi: 10.3389/fchem.2014.00093, the contents are herebyincorporated by reference in their entirety for all purposes.

One or more of the adiponectin peptidomimetic compound described hereincan be included in a therapeutically effective composition such as anophthalmic composition. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9,10 or more different adiponectin peptidomimetic compound can becontained in the composition. The amounts of each adiponectinpeptidomimetic compound can be the same (or equal) or different. Theactivity of each adiponectin peptidomimetic compound can be the same ordifferent.

1. Linking Groups (M₁ and M₂)

The linking group (M₁ or M₂) for coupling X or Z in a compound ofFormula I may be any moiety that is at least bifunctional, provided thatthe resulting link between X or Z and the N-terminal or C-terminal aminoacid or non-natural amino acid is stable. Suitable linking groupsinclude bi- and multi-functional alkyl, aryl, aralkyl or peptidicmoieties, alkyl, aryl or aralkyl aldehydes acids esters and anhydrides,sulfhydryl or carboxyl groups, such as maleimido benzoic acidderivatives, maleimido propionic acid derivatives and succinimidoderivatives or may be derived from cyanuric bromide or chloride,carbonyldiimidazole, succinimidyl esters or sulphonic halides and thelike (Fischer et al., U.S. Pat. No. 6,472,507, the entire disclosure ofwhich is incorporated herein by reference). The functional groups on thelinker moiety may include amino, hydrazino, hydroxyl, thiol, maleimido,carbonyl, and carboxyl groups.

Optionally the linker group is selected so as to be sufficiently labile(e.g., to enzymatic cleavage by an enzyme present in the targetedtissue) so that it is cleaved following transport of a peptide of theinvention, thereby releasing the peptide. Exemplary labile linkages aredescribed in Low et al., U.S. Pat. No. 5,108,921, the entire disclosureof which is incorporated herein by reference. The peptide-active agentdelivery system may also dissociate by way of chemical cleavage betweenthe active agent and peptide of the invention. Within the embodimentswherein the linker moiety includes amino acid residues, such cleavagemay occur within the linker moiety itself.

If the link formed by the linking group M₁ is between the Xaai and acarboxyl group of X (for example if X is a tagging element, the terminalcarboxyl group of a peptidic tagging element or the terminal carboxylgroup of a molecule), any amino acid (including, but not restricted to,.alpha.-amino acids including, but not restricted to, the proteinogenicamino acids) or peptide chain may form the link between Xaai and X.

In some embodiments, if the bonds between the M₁ or M₂ group and thepeptides are amide bonds, the link may be formed by means of anyfunctional groups capable of forming bonds between the Xaai or Xaa₅ anda —C(═0)-group of the terminal (or other) carboxyl group (or theterminal, or other —NH— group, or any other functional group of X or Zrespectively). In other embodiments, if the link formed by the linkinggroup M₁ is between the Xaai and an amino or carboxyl group of X, anyamino acid or peptide chain may form the link between Xaai and X. Inother embodiments, if the link formed by the linking group M2 is betweenthe Xaa₅ and an amino or carboxyl group of Z, any amino acid or peptidechain may form the link between Xaa₅ and Z. Examples of suitable linkinggroups MI and M2 for connecting, for instance, the Xaai and a carboxylgroup of X, the Xaai and an amino group of X, the Xaa₅ and a carboxylgroup of Z, and the Xaa₅ and an amino group of Z, are described in U.S.Pat. App. Publication No. 2014/0057833, the disclosure of which ishereby incorporated in its entirety for all purposes.

2. Peptide Transduction Domain

In some embodiments, X and/or Z may comprise a protein transductiondomain. A protein transduction domain includes a peptide that is capableof crossing cell membranes and of directing the transport of a peptide,protein, or molecule associated with the protein transduction domain;from the outside of a cell into the cytoplasm of the cell through thecytoplasmic membrane of the cell. In some instances, the proteintransduction domain comprises a relatively short sequence derived from anaturally occurring protein, such as the TAT protein of HIV, theantennapedia protein from Drosophila, and the VP22 protein from theherpes simplex virus. Non-limiting examples of suitable proteintransduction domains are described in, e.g., Handbook ofCell-Penetrating Peptides, by Ulo Langel (Editor) (CRC Press, 2ndEdition, 2006). Cell-Penetrating Peptides: Process and Applications, byUlo Langel (Editor) (CRC Press, I .sup.st Edition, 2002); E. L. Snyder,et al, Pharm. Res., 2004, 21(3), 389-93. Beerens et al., Current GeneTherapy, 2003, 3(5), 486-94; Hudec et al., Med. Res. Rev., 2005, 25(6),679-736. Detailed descriptions of useful protein transduction domainsare found in, e.g., U.S. Pat. App. Publication No. 2014/0057833, thedisclosure of which is hereby incorporated in its entirety for allpurposes.

3. Preparing Adiponectin Peptidomimetic Compounds

Peptidomimetic compounds of the present invention may be naturalpeptides, recombinant peptides or synthetic peptides. They may also bechemically synthesized, using, for example, solid phase synthesismethods. Additionally, peptide transduction domains appended to peptidesof the invention may be natural or synthetic peptides, and may be eitherprepared by isolation from natural sources or may be synthesized.

The peptides of the present invention may be synthesized de novo usingpeptide synthesis methods. In such methods. the peptide chain isprepared by a series of coupling reactions in which the constituentamino acids are added to the growing peptide chain in the desiredsequence. The use of various N-protecting groups, e.g., thecarbobenzyloxy group or the t-butyloxycarbonyl group; various couplingreagents e.g., dicyclohexylcarbodiimide or carbonyldiimidazole; variousactive esters, e.g., esters of N-hydroxyphthalimide orN-hydroxy-succinimide; and the various cleavage reagents, e.g.,trifluoroacetic acid (TFA), HCl in dioxane, boron tris-(trifluoracetate)and cyanogen bromide; and reaction in solution with isolation andpurification of intermediates are methods well-known to those ofordinary skill in the art. The reaction may be carried out with thepeptide either in solution or attached to a solid phase support. In thesolid phase method, the peptide is released from the solid phase supportfollowing completion of the synthesis.

In some embodiments, the peptide synthesis method may follow Merrifieldsolid-phase procedures. See, e.g., Merrifield, J. Am. Chem. Soc, 1963.85, 2149-54. Additional information about the solid phase syntheticprocedure can be obtained from, for example, Solid Phase PeptideSynthesis: A Practical Approach by E. Atherton and R. C. Sheppard(Oxford University Press. 1989, Solid phase peptide synthesis, by J. M.Stewart and J. D. Young, (2nd edition, Pierce Chemical Company.Rockford, 1984), and the review chapters by R. Merrifield in Advances inEnzymology 32:221-296, edited by F. F. Nold (Interscience Publishers,New York, 1969) and by B. W. Erickson and R. Merrifield in The ProteinsVol. 2, pp. 255 et seq., edited by Neurath and Hill, (Academic Press,New York, 1976). Peptide synthesis may follow synthetic techniques suchas those set forth in Fields et al., Introduction to Peptide Synthesis,in Current Protocols in Molecular Biology (Chapter 11, Unit 11.15; JohnWiley and Sons, 2008) and Amblard et al. (2006, Molecular Biotechnology,33:239-254).

The synthesis of peptides by solution methods is described in, forexample, The Proteins, Vol. 11, edited by Neurath et al. (3rd Edition,Academic Press 1976). Other general references to the synthesis ofpeptides include: Peptide Synthesis Protocols, edited by M. W.Pennington and Ben M. Dunn (Humana Press 1994), Principles of PeptideSynthesis, by Miklos Bodanszky (2nd edition, Springer-Verlag, 1993), andChemical Approaches to the Synthesis of Peptides and Proteins by PaulLloyd-Williams, F. Albericio, E. Giralt (CRC Press 1997), and SyntheticPeptides: A User's Guide, edited by G. Grant (Oxford University Press,2002).

Alternatively, peptides may be prepared utilizing recombinant DNAtechnology, which comprises combining a nucleic acid encoding peptidesof Formula I or II in a suitable vector, inserting the resulting vectorinto a suitable host cell, recovering the peptide subsequently producedby the host cell, and purifying the polypeptide recovered. The requiredtechniques of recombinant DNA and protein technology are known to theordinary skilled artisan. General methods for the cloning and expressionof recombinant molecules are described in Molecular Cloning by Sambrooket al. (Cold Spring Harbor Laboratories, Second Ed., 1989) and inCurrent Protocols in Molecular Biology by Ausubel (Wiley and Sons,1987).

The nucleic acid encoding a desired peptide may be operatively linked toone or more regulatory regions. Regulatory regions include promoters,polyadenylation signals, translation initiation signals (Kozak regions),termination codons, peptide cleavage sites, and enhancers. Theregulatory sequences used must be functional within the cells of thevertebrate in which they are administered. Selection of the appropriateregulatory region or regions is a routine matter, within the level ofordinary skill in the art.

The compounds of the invention, whether prepared by chemical synthesisor recombinant DNA technology, may be purified using known techniques,for example preparative HPLC, FPLC, affinity chromatography, as well asother chromatographic methods. Isolated compounds may then be assessedfor biological activity according to the methods described herein, aswell as by any methods known to the skilled artisan.

For synthetic techniques, peptides can be produced by the establishedprocedure of solid phase peptide synthesis. Briefly, this procedureentails the sequential assembly of the appropriate amino acids into apeptide of a desired sequence while the end of the growing peptide islinked to an insoluble support. Usually, the carboxyl terminus of thepeptide is linked to a polymer from which it can be liberated upontreatment with a cleavage reagent.

The amino acid Xaai, Xaa₅ or Xaa₄ when Xaa₅ is zero amino acid may beconjugated to a lipophilic compound comprising X or Z either directly orby use of a linker. The lipophilic compound may be a natural compoundsuch as a saturated or unsaturated fatty acid, a fatty acid diketone, aterpene, a prostaglandin, a vitamin; a carotenoid or steroid or asynthetic compound such as a carbon acid, an alcohol, an amine andsulphonic acid with one or more alkyl-, aryl-, alkenyl-, or othermultiple unsaturated compounds. The conjugation between the amino acidand the lipophilic compound, optionally through a linker may be doneaccording to methods known in the art, e.g. as described by Bodanszky inPeptide Synthesis. John Wiley. New York, 1976 and in WO 96/12505.

To covalently attach a polymer molecule to an amino acid of the compounddescribed herein, the hydroxyl end groups of the polymer molecule mustbe provided in activated form, i.e., with reactive functional groups(for example primary amino groups, hydrazide (HZ), thiol, succinate(SUC), succinimidyl succinate (SS), succinimidyl succinamide (SSA),succinimidyl proprionate (SPA), succinimidyl carboxy methyl ate (SCM),benzotriazole carbonate (BTC), N-hydroxysuccinimide (NHS), aldehyde,nitrophenyl carbonate (NPC), and tresylate (TRES). Specific examples ofactivated PEG polymers include the following linear PEGs: NETS-PEG (e.g.SPA-PEG, succinimidyl succinate proprionate-PEG (S SPA-PEG), SBA-PEG,SS-PEG, SSA-PEG, succinimidyl carbonate-PEG (SC-PEG), succinimidylglutarate-PEG (SG-PEG), and SCM-PEG and NOR-PEG), BTC-PEG, epoxide-PEG(EPDX-PEG), isocyanate-PEG (NCO-PEG), NPC-PEG, carbonylimidazole-PEG(CDI-PEG), aldehyde-PEG (ALD-PEG), TRES-PEG, VS-PEG, iodo-PEG, andmaleimide-PEG (MAL-PEG), and branched PEGs such as PEG2-NHS and thosedisclosed in U.S. Pat. Nos. 5,932,462 and 5,643,575, both of whichreferences are hereby incorporated by reference in their entirety. ThePEGylation may be directed towards specific attachment groups, e.g., theN-terminal amino group (U.S. Pat. No. 5,985,265). Furthermore, theconjugation may be achieved in one step or in a stepwise manner (e.g. asdescribed in WO 99/55377).

B. Ocular Disorders Amenable to Treatment with AdiponectinPeptidomimetic Compounds

The adiponectin peptidomimetic compounds described herein can be used totreat an ocular disease or disorder, including dry eye or an oculardisease associated with inflammation.

Dry eye disease or keratoconjunctivitis sicca (KCS) can be caused by orassociated with various conditions including, but not limited to Sjogrensyndrome, ocular cicatrical pemphigoid, congenital alacrima,blepharitis, lacrimal gland ablation, age-related lacrimal glanddeficiency, alacrima (e.g., Triple A or Allgrove syndrome, and Riley-Daysyndrome), lacrimal gland infiltration (e.g., sarcoidosis, lymphoma, andAIDS), lacrimal gland duct obstruction, meibomian gland disorder,pterygium, chronic inflammation of the conjunctiva, reflex block, herpeszoster, ocular allergies, autoimmune disease, chronic graft-versus-hostdisease, the natural aging process, diabetes, long-term contact lenswear, dry environment, excessive computer screen use, surgery thatinvolves corneal incisions or ablates corneal nerves (e.g., cataractsurgery, refractive surgery, retinal surgery, ocular tumor therapy,medications, decreased blinking (low blink rate), disorders of lidaperature or lid/globe dynamics, pregnancy, polycystic ovary syndrome,acne rosacea, lupus, scleraderma, sarcoidosis, Stevens-Johnson syndrome,Parkinson's disease, thyroid disease, cosmetic surgery, smoking,radiation therapy, vitamin A deficiency, and menopause.

Dry eye can also be caused by nutritional disorders and deficiencies,pharmacologic side effects, skin disease on and around the eyelids, eyestress and glandular and tissue destruction, environmental exposure tosmog, smoke, excessively dry air, airborne particulates, autoimmune andother immunodeficient disorders. In dry eye, the ocular surfaceepithelium undergoes squamous metaplasia, manifested by loss of gobletcells, mucin deficiency and keratinization. These changes result in tearfilm instability, which leads to the clinical symptoms of dry eyesyndrome.

Symptoms of dry eye include stinging, burning or scratchy sensation inthe eye; ocular dryness or grittiness; stringy mucus in or around theeye; increase eye irritation; eye fatigue; sensitivity to light(photophobia); eye redness; excessive tearing; episode of blurredvision; foreign body sensation in the eye; pain or soreness around or inthe eye; inability to cry when emotionally stressed; decreased toleranceof an activity requiring sustained visual attention; and any combinationthereof. Symptoms of dry eye can be quantified using, for example, inthe Ocular Surface Disease Index (OSDI) questionnaire, which lists 12symptoms and grades each on a scale of 1-4. Clinical signs of dry eyecan be assessed, for example, by performing impression cytology (e.g.,ocular surface staining), measuring tear breakup time (TBUT), performingthe Schirmer's test, performing a phenol red thread tear test, andmeasuring the components of tears (e.g., analysis of tear proteins ortear-film osmolarity). Elevated osmolarity (hyperosmolarity) may causeless regulation of tear film, more damage to the ocular surface, and insome cases, increased inflammation of the eye.

Ocular diseases associated with inflammation include, but are notlimited to, uveitis, dry eye, keratitis, allergic eye disease,infectious keratitis, herpetic keratitis, corneal angiogenesis,lymphangiogenesis, uveitis, pterygium, retinitis, choroiditis, acutemultifocal placoid pigment epitheliopathy, Behcet's disease,post-surgical corneal wound healing, conditions caused by laser,conditions caused by photodynamic therapy, wet and dry age-relatedmacular degeneration (ARMD), conditions affecting the posterior part ofthe eye, maculopathies, retinal degeneration, non-exudative age relatedmacular degeneration, exudative age related macular degeneration,choroidal neovascularization, diabetic retinopathy (proliferative),retinopathy of prematurity (ROP), acute macular neuroretinopathy,central serous chorioretinopathy, cystoid macular edema, and diabeticmacular edema, birdshot retinochoroidopathy, infectious (syphilis, lyme,tuberculosis, toxoplasmosis), intermediate uveitis (pars planitis),multifocal choroiditis, multiple evanescent white dot syndrome (mewds),ocular sarcoidosis, posterior scleritis, serpiginous choroiditis,subretinal fibrosis and uveitis syndrome, Vogt-Koyanagi and Haradasyndrome, retinal arterial occlusive disease, central retinal veinocclusion, cystoids macular edema, disseminated intravascularcoagulopathy, branch retinal vein occlusion, hypertensive funduschanges, ocular ischemic syndrome, retinal arterial microaneurysms,Coat's disease, parafoveal telangiectasis, hemi-retinal vein occlusion,papillophlebitis, central retinal artery occlusion, branch retinalartery occlusion, carotid artery disease (CAD), frosted branch angiitis,sickle cell retinopathy and other hemoglobinopathies, angioid streaks,familial exudative vitreoretinopathy, and Eales disease, sympatheticophthalmia, uveitic retinal disease, retinal detachment, trauma,conditions caused by photodynamic therapy, photocoagulation,hypoperfusion during surgery, radiation retinopathy, and bone marrowtransplant retinopathy, proliferative vitreal retinopathy and epiretinalmembranes, proliferative diabetic retinopathy, ocular histoplasmosis,ocular toxocariasis, presumed ocular histoplasmosis syndrome (PONS),endophthalmitis, toxoplasmosis, retinal diseases associated with HIVinfection, choroidal disease associate with HIV infection, uveiticdisease associate with HIV infection, viral retinitis, acute retinalnecrosis, progressive outer retinal necrosis, fungal retinal diseases,ocular syphilis, ocular tuberculosis, diffuse unilateral subacuteneuroretinitis, myiasis, retinitis pigmentosa, systemic disorders withaccosiated retinal dystrophies, congenital stationary night blindness,cone dystrophies, Stargardt's disease, fundus flavimaculatus, Best'sdisease, pattern dystrophy of the retinal pigmented epithelium, X-linkedretinoschisis, Sorsby's fundus dystrophy, benign concentric maculopathy,Bietti's crystalline dystrophy, pseudoxanthoma elasticum; retinaldetachment, macular hole, giant retinal tear, retinal disease associatedwith tumors, congenital hypertrophy of the retinal pigmented epithelium,posterior uveal melanoma, choroidal hemangioma, choroidal osteoma,choroidal metastasis, combined hamartoma of the retina and retinalpigmented epithelium, retinoblastoma, vasoproliferative tumors of theocular fundus, retinal astrocytoma, intraocular lymphoid tumors, otherdiseases affecting the posterior part of the eye, punctate innerchoroidopathy, acute posterior multifocal placoid pigmentepitheliopathy, myopic retinal degeneration, acute retinal pigmentepitheliitis, postsurgical corneal inflammation, cornealtransplantation, blepharitis, MGD, glaucoma, ocular hypertension, branchvein occlusion, retinal diseases, Best's vitelliform maculardegeneration, retinitis pigmentosa, proliferative vitreoretinopathy(PVR), and any other degenerative diseases of either the photoreceptorsor the retinal pigment epithelial (RPE).

Symptoms or clinical signs of ocular diseases associated withinflammation include, but are not limited to, unstable tear film,chronic hyperosmolar stress, evaporative tear loss, decreased lubricity,other tear deficiencies that lead to an increased in pro-inflammatoryresponse in the eye, inflammation, and any combination thereof.

C. Pharmaceutical Compositions

The adiponectin peptidomimetic compounds can be used and formulated intoany of a number of pharmaceutical compositions, including thosedescribed in the United States Pharmacopeia (U.S.P.), Goodman andGilman's The Pharmacological Basis of Therapeutics, IO.sup.th Ed.,McGraw Hill, 2001; Katzung, Ed., Basic and Clinical Pharmacology,McGraw-Hill/Appleton & Lange, 8th ed., Sep. 21, 2000; Physician's DeskReference (Thomson Publishing; and/or The Merck Manual of Diagnosis andTherapy, 18th ed., 2006, Beers and Berkow, Eds., Merck Publishing Group;or, in the case of animals, The Merck Veterinary Manual, 9th ed., KahnEd., Merck Publishing Group, 2005.

Ophthalmic pharmaceutical compositions may also contain one or moreexcipients or other substances, such as preservatives, antioxidants, pHadjusting agents, buffering agents, gelling agents, viscosity enhancers,surfactants, solubility agents, lubricating agents, salts, co-solvents,diluents, carriers, adjuvants, oils, humectants, emollients,stabilizers, emulsifying agents, and/or dispersing agents. Other agentsmay be employed in the compositions for a variety of purposes. By way ofexample, injectable compositions may contain various excipients or othersubstances, such as preservatives, antioxidants, pH adjusting agents,buffering agents, salts, emulsifying agents, and/or dispersing agents.Non-limiting examples of a preservative such as a water-solublepreservative include sodium bisulfite, sodium bisulfate, sodiumthiosulfate, benzalkonium chloride, chlorobutanol, thimerosal, ethylalcohol, methylparaben, polyvinyl alcohol, benzyl alcohol, andphenylethyl alcohol. Examples of ophthalmically acceptable antioxidantsinclude, but are not limited to, sodium bisultite, sodium thiosulfate,acetyl cysteine, cysteine, thioglycerol, sodium sulfite, acetone sodiumbisulfite, dithioerythreitol, dithiothreitol, thiourea, and erythorbicacid. Useful examples of ophthalmically acceptable pH adjusting agents,such as an acid, base and/or buffer include but are not limited to, anacid such as acetic, boric, citric, lactic, phosphoric, sulfuric, andhydrochloric acids; a base such as sodium hydroxide, sodium phosphate,sodium borate, sodium citrate, sodium acetate, sodium lactate andtris-hydroxymethylarainomethane, triethanolamine; and/or a buffer suchas citrate/dextrose, sodium bicarbonate and ammonium chloride or anamino acid. Such an acid, base and/or buffer can be included in anamount sufficient to adjust pH of the composition to an ophthalmicallyacceptable range.

The composition can be formulated for topical ophthalmic application,for example, in the form of solutions, ointments, creams, lotions, eyeointments and, most preferably, eye drops or eye gels and can containthe appropriate conventional additives, including, for example,preservatives, solvents to assist drug penetration, and emollients inointments and creams. The ophthalmic vehicles include, but are notlimited to, saline solution, water polyethers such as polyethyleneglycol, polyvinyls such as polyvinyl alcohol and povidone, cellulosederivatives such as methylcellulose and hydroxypropyl methylcellulose,petroleum derivatives such as mineral oil and white petrolatum, animalfats such as lanolin, polymers of acrylic acid such ascarboxypolymethylene gel, vegetable fats such as peanut oil andpolysaccharides such as dextrans, and glycosaminoglycans such as sodiumhyaluronate and salts such as sodium chloride and potassium chloride.

Solutions of the active compounds as free base or pharmacologicallyacceptable salt can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations can contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical compositions containing invention compounds may be in aform suitable for topical use, for example, as oily suspensions, assolutions or suspensions in aqueous liquids or nonaqueous liquids, or asoil-in-water or water-in-oil liquid emulsions. Alternatively, thecompounds can be formulated for injection into the eye, such asintravitreal injection subj conjunctival injection and injection intothe anterior chamber of the eye. In other instances, the compounds maybe in a form suitable for implantation use, e.g., as entrapped inmicrocapsules. The active ingredients may also be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).The compositions can be also in an ophthalmic depot formulation, such asfor subconjunctival administration. The adiponectin peptidomimetics canbe embedded in in a biocompatible pharmaceutically acceptable polymer ora lipid encapsulating agent. The depot formulations may be adapted torelease all or substantially all the active material over an extendedperiod of time. The polymer or lipid matrix, if present, may be adaptedto degrade sufficiently to be transported from the site ofadministration after release of all or substantially all the activeagent. The depot formulation can be a liquid formulation, comprising apharmaceutical acceptable polymer and a dissolved or dispersed activeagent, upon injection, the polymer forms a depot at the injections site,e.g., by gelifying or precipitating. The composition can comprise asolid article that can be inserted in a suitable location in the eye,such as between the eye and eyelid or in the conjuctival sac, where thearticle releases the active agent. Solid articles suitable forimplantation in the eye in such fashion generally comprise polymers andcan be bioerodible or non-bioerodible.

Pharmaceutical compositions may be prepared by combining atherapeutically effective amount of at least one compound according tothe present invention, or a pharmaceutically acceptable salt thereof, asan active ingredient with conventional ophthalmically acceptablepharmaceutical excipients and by preparation of unit dosage suitable forocular use. The therapeutically efficient amount typically is betweenabout 0.0001% (wt) and about 90% (wt), preferably about 0.0001% (wt) toabout 50% (wt) in liquid formulations.

Alternatively, the active compounds may be applied to the eye vialiposomes. Further, the active compounds may be infused into the tearfilm via a pump-catheter system. In some embodiments, the activecompound is contained within a continuous or selective-release device,for example, membranes such as, but not limited to, those employed inthe pilocarpine (Ocusert™) System (Alza Corp., Palo Alto, Calif). Insome embodiments, the active compounds is contained within, carried by,or attached to contact lenses which are placed on the eye. In otherembodiments, the active compound is contained within a swab or spongewhich can be applied to the ocular surface. In another embodiment, theactive compound is contained within a liquid spray which can be appliedto the ocular surface. In another embodiment, the active compound isinjected directly into the lacrimal tissues or onto the eye surface.

When the pharmaceutical composition for treating dry eye is used as anophthalmic solution, it is provided in any dosage form which is used forophthalmic solution, for example, an aqueous eye drop such as aqueousophthalmic solution, aqueous suspended ophthalmic solution, viscousophthalmic solution and solubilized ophthalmic solution, or anon-aqueous ophthalmic solution such as non-aqueous ophthalmic solutionand non-aqueous suspended ophthalmic solution. Among these, the aqueousophthalmic solution is preferable.

When the pharmaceutical composition for treating dry eye is preparedinto an aqueous ophthalmic solution, various additives normally used inthe aqueous ophthalmic solution are conveniently contained therein aslong as the object of the present invention is not adversely affected.Examples of such the additives include buffers, isotonizing agents,preservatives, solubilizers (stabilizers), pH adjusting agents,osmolarity adjusting agents, thickeners and chelating agents.

The buffers may be selected from, but not limited to, the groupcomprising a phosphate buffer, a borate buffer, a citrate buffer, atartrate buffer, an acetate buffer (for example, sodium acetate) and anamino acid. The isotonizing agents may be selected from, but not limitedto, the group comprising sugars such as sorbitol, glucose and mannitol,polyhydric alcohols such as glycerin, polyethylene glycol andpolypropylene glycol, and salts such as sodium chloride. Thepreservatives may be selected from, but not limited to, the groupcomprising benzalkonium chloride, benzethonium chloride, alkylparaoxybenzoates such as methyl paraoxybenzoate and ethylparaoxybenzoate, benzyl alcohol, phenethyl alcohol, sorbic acid andsalts thereof, thimerosal and chlorobutanol. The solubilizers(stabilizers) may be selected from, but not limited to, the groupcomprising cyclodextrin and derivatives thereof, water-soluble polymerssuch as polyvinylpyrrolidone), and surfactants such as polysorbate 80(trade name: Tween 80). The pH adjusting agents may be selected from,but not limited to, the group comprising hydrochloric acid, acetic acid,phosphoric acid, sodium hydroxide, potassium hydroxide and ammoniumhydroxide. The thickeners may be selected from, but not limited to, thegroup comprising hydroxyethylcellulose, hydroxypropylcellulose,methylcellulose, hydroxypropylmethylcellulose and carboxymethylcelluloseand salts thereof. The chelating agents may be selected from, but notlimited to, the group comprising sodium edetate, sodium citrate andsodium condensed phosphate.

For ophthalmic application, preferably solutions are prepared using aphysiological saline solution as a major vehicle. The pH of suchophthalmic solutions should preferably be maintained between 4.5 and8.0; about 5 to 7.5; preferably 6 to 7 with an appropriate buffersystem, a neutral pH being preferred but not essential. Examples ofacids include acetic, boric, citric, lactic, phosphoric, hydrochloric,and the like, and examples of bases include sodium hydroxide, sodiumphosphate, sodium borate, sodium citrate, sodium acetate, sodiumlactate, tromethamine, THAM (trishydroxymethylamino-methane), and thelike. Salts and buffers include citrate/dextrose, sodium bicarbonate,ammonium chloride and mixtures of the aforementioned acids and bases.

The osmotic pressure of the aqueous ophthalmic composition is generallyfrom about 200 to about 400 milliosmolar (mOsM), more preferably from260 to 340 mOsM. The osmotic pressure can be adjusted by usingappropriate amounts of physiologically and ophthamologically acceptableionic or non-ionic agents. Sodium chloride is a preferred ionic agent,and the amount of sodium chloride ranges from about 0.01% to about 1%(w/v), and preferably from about 0.05% to about 0.45% (w/v). Equivalentamounts of one or more salts made up of cations such as potassium,ammonium and the like and anions such as chloride, citrate, ascorbate,borate, phosphate, bicarbonate, sulfate, thiosulfate, bisulfate, sodiumbisulfate, ammonium sulfate, and the like can be used in addition to orinstead of sodium chloride to achieve osmolality within the above-statedrange. Further, non-ionic agents such as mannitol, dextrose, sorbitol,glucose and the like can also be used to adjust the osmolality.

Tonicity adjusters may be added as needed or convenient. They include,but are not limited to, salts, particularly sodium chloride, potassiumchloride, mannitol and glycerin, or any other suitable ophthalmicallyacceptable tonicity adjuster. Various buffers and means for adjusting pHmay be used so long as the resulting preparation is ophthalmicallyacceptable. Accordingly, buffers include acetate buffers, citratebuffers, phosphate buffers and borate buffers. An ophthalmicallyacceptable antioxidant for use in the present invention includes, but isnot limited to, sodium metabi sulfite, sodium thiosulfate,acetylcysteine, butylated hydroxyanisole and butylated hydroxytoluene.

When the pharmaceutical composition for treating dry eye is preparedinto an ophthalmic ointment, a base compound must be present. The baseof the ophthalmic ointment may be selected from but not limited by thegroup comprising purified lanolin, VASELINE® plastibase, liquid paraffinand polyethylene glycol.

The formulations may also contain conventional pharmaceuticallyacceptable preservatives, stabilizers and surfactants. Preferredpreservatives that may be used in the pharmaceutical compositions of thepresent invention include, but are not limited to, benzalkoniumchloride, chlorobutanol, thimerosal, phenylmercuric acetate andphenylmercuric nitrate. A preferred surfactant is, for example, Tween80. Likewise, various preferred vehicles may be used in the ophthalmicpreparations of the present invention. These vehicles include, but arenot limited to, polyvinyl alcohol, povidone, hydroxypropyl methylcellulose, poloxamers, carboxymethyl cellulose, hydroxyethyl cellulosecyclodextrin and purified water.

Alternatively, the composition of the invention can be formulated fororal administration using pharmaceutically acceptable tabletingexcipients including lactose, microcrystalline cellulose, corn starch,stearic acid, or the like, can be used. Oral administration can alsocomprise a liquid composition formulated in water, glycols, oils,alcohols or the like.

The adiponectin peptidomimetic compounds can be formulated intolipid-based nanocarriers, such as solid lipid nanoparticles,nanostructured lipid carriers, lipid-drug conjugates, andcoated-liposomes.

The formulations of the present invention are conveniently packaged informs suitable for metered application, such as in containers equippedwith a dropper, to facilitate application to the eye. Containerssuitable for dropwise application are usually made of suitable inert,non-toxic plastic material, and generally contain between about 0.5 andabout 15 ml solution. One package may contain one or more unit doses.Especially preservative-free solutions are often formulated innon-resealable containers containing up to about ten, preferably up toabout five units doses, where a typical unit dose is from one to about 8drops, preferably one to about 3 drops. The volume of one drop usuallyis about 20-35 μl.

The formulations may be in the form of a sterile solution or suspension.The solution or suspension can be for topical or injectable application.It can be in a sterile injectable formulation, e.g., a liquid orsuspension formulation. In some embodiments, it may be formulatedaccording to known methods using suitable dispersing or wetting agentsand suspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic intraocularly-or intravitreally-acceptable diluent or solvent. Buffers, preservatives,antioxidants, and the like can also be incorporated as required.

Provided herein are kits comprising the ophthalmic composition orformulation described herein. In some embodiments, the kit also includesinstructions for administering the composition or formulation. In somecases, the composition or formulation is packaged in a form suitable formetered application. In other cases, the composition or formulation ispackaged for single unit dose use.

D. Methods of Administration

The composition provided herein can be administered to the eye. In someembodiments, the composition is applied to the palpebral part of theeye, such as the external portion of the upper and lower eyelids and themedial and lateral canthus, and/or the ocular surface of the eye. Insome instances, the compositions can be administered to an afflicted eyeconjunctival sac. In some embodiments, the composition is administeredtopically, by intravitreal injection, by subconjunctival injection, byconjunctival injection, by intramuscular injection, by subcutaneousinjection, by intravenous injection, by intracameral injection, or byimplantation into the subject's eye. In some cases, administrationincludes intravitreal depot implantation or other ophthalmic drugdelivery methods described in, e.g., Edelhauser et al., InvestOphthalmol Vis Sci, 2010, 51(11):5403-5420. The eye includes, but is notlimited to, a tissue, gland, vessel, lens, muscle, nerve, or otherstructure in or around the eye such as an ocular tissue, ocular surface,ocular chamber, eyelid, nasolacrimal duct, meibomian gland, and lacrimalgland.

The composition can be formulated for ophthalmic application, forexample, in the form of solutions, ointments, creams, lotions, eyeointments and, most preferably, eye drops or eye gels and can containthe appropriate conventional additives, including, for example,preservatives, solvents to assist drug penetration, and emollients inointments and creams. Such topical formulations can contain compatibleconventional carriers, for example cream or ointment bases, and ethanolor oleyl alcohol for lotions.

The composition can be administered to the eyes of a patient by anysuitable means, but are preferably administered as a liquid or gelsuspension in the form of drops, spray or gel. In one embodiment, theformulation is in the form of drops, and is dropped onto the ocularsurface. In another embodiment, the formulation is contained within aswab or sponge which can be applied to the ocular surface. In anotherembodiment, the formulation is contained within a liquid spray orointment which can be applied to the ocular surface. In anotherembodiment, the formulation is injected directly into the eye, such asinto the lacrimal tissues or onto the eye surface. In a furtherembodiment, the formulation is first applied on a fingertip or otherapplicator, then applied or rubbed directly onto the lid margin orcanthus. Alternatively, the adiponectin peptidomimetic compound can beapplied to the eye via a colloidal dosage form such as nanoparticular,nanomicelles, liposomes, and microemulsions. Further, the compositioncan be infused into the tear film via a pump-catheter system. Anotherembodiment involves the adiponectin peptidomimetic compound containedwithin a continuous or selective-release device, for example, membranes.As an additional embodiment, the adiponectin peptidomimetic compound canbe contained within, carried by, or attached to contact lenses or othercompatible controlled release materials, which are placed on the eye oraround the eye.

In some embodiments, the compositions are administered topically,intraocularly, intracamerally, intraorbitally, perio-ocularly,intravitreally, subconjunctivally, conjunctivally, intramuscularly,subcutaneously, intravenously, intracamerally, or via other routes in oraround the eye. Non-limiting delivery routes for the therapeuticcompositions described herein include aqueous solution, oily solutions,e.g., ointments, colloidal carriers, e.g., micelles, emulsions,liposomes, nanoparticles, solids forms, e.g., collagen-based shieldsand/or particles, and drug-loaded punctual plugs, drug-loadedcanalicular plugs, contact lenses, implants and inserts.

The suitability of a particular route of administration will depend inpart on the pharmaceutical composition, its components, the disorderbeing treated, and the subject in need of the therapy.

E. Dosing

The dosage of a therapeutic agent administered to a subject will varydepending on a wide range of factors. For example, it would be necessaryto provide substantially larger doses to humans than to smaller animals.The dosage will depend upon the size, age, sex, weight, medical historyand condition of the subject, and the nature of the dry eye diseasebeing treated, use of other therapies, the potency of the substancebeing administered, and the frequency of administration. The skilledartisan will be able to determine appropriate dosages depending on theseand other factors. For example, when the therapeutic agent can be usedas an ophthalmic solution for treating dry eye in a subject in needthereof, it can be desirable that the aqueous solution eye drop containthe therapeutic agent in an amount of about 0.001% (wt) to 30% (wt),e.g, about 0.001% (wt), about 0.005% (wt), about 0.01% (wt), about 0.02%(wt), about 0.03% (wt), about 0.04% (wt), about 0.05% (wt), about 0.06%(wt), about 0.07% (wt), about 0.08% (wt), about 0.09% (wt), about 0.1%(wt), about 0.2% (wt), about 0.3% (wt), about 0.4% (wt), about 0.5%(wt), about 0.6% (wt), about 0.7% (wt), about 0.8% (wt), about 0.9%(wt), about 1% (wt), about 2% (wt), about 3% (wt), about 4% (wt), about5% (wt), about 6% (wt), about 7% (wt), about 8% (wt), about 9% (wt),about 10% (wt), about 11% (wt), about 12% (wt), about 13% (wt), about14% (wt), about 15% (wt), about 16% (wt), about 17% (wt), about 18%(wt), about 19% (wt), about 20% (wt), about 21% (wt), about 22% (wt),about 23% (wt), about 24% (wt), about 25% (wt), about 26% (wt), about27% (wt), about 28% (wt), about 29% (wt), about or 30% (wt). In someembodiments, the therapeutic agent can be in an amount ranging fromabout 0.001% (wt) to about 30% (wt), about 0.005% (wt) to about 30%(wt), about 0.01% (wt) to about 30% (wt), about 0.1% (wt) to about 30%(wt), about 1% (wt) to about 30% (wt), about 10% (wt) to about 30% (wt),about 20% (wt) to about 30% (wt), about 10% (wt) to about 20% (wt),about 0.001% (wt) to about 10% (wt), about 0.001% (wt) to about 1% (wt),about 0.001% (wt) to about 0.1% (wt), 0.01% (wt) to about 0.1% (wt), andthe like. When administered, the therapeutic agent can be given oncedaily or with multiple daily doses such as twice per day, three timesper day and four times per day. In some embodiments, the therapeuticagent can be administered once a day, every other day, or lessfrequently. The therapeutic agent can be administered when the subjecthas one or more symptoms of dry eye or an ocular disease. In someinstances, the therapeutic agent can be given in a dose of one to fivedrops or more, for example, one drop, two drops, three drops, fourdrops, five drops or more. When administered, the compositions may begiven once daily or with multiple daily doses such as twice per day,three times per day four times per day, five times per day or more. Insome embodiments, the therapeutic agent can be administered lessfrequently then once daily. For instance, the therapeutic agent can beadministered every week, every 2 weeks, every 3 weeks, every 4 weeks,every 6 weeks, every 7 weeks, every 8 weeks, or less frequently. In someembodiments, the therapeutic agent can be administered according to theseverity of the symptoms experienced by the subject.

Lifitegrast when administered topically, e.g., as eye drops orointments, or for intraorbital or perio-ocular injection, exemplarydosages can be from about 0.1 mg/ml to about 200 mg/ml.

An effective amount of an adiponectin peptidomimetic compound willdepend on the age, sex and weight of the patient, the current medicalcondition of the patient and the nature of the dry eye disease beingtreated. The skilled artisan will be able to determine appropriatedosages depending on these and other factors. For example, when thepharmaceutical composition is used as an ophthalmic solution fortreating dry eye in a subject in need thereof, it is desirable that theaqueous solution eye drop contain the adiponectin peptidomimeticcompound in an amount of about 0.0001% (wt) to 90% (wt), e.g., about0.0001% (wt), about 0.0005% (wt),about 0.001% (wt), about 0.005% (wt),about 0.01% (wt), about 0.02% (wt), about 0.03% (wt), about 0.04% (wt),about 0.05% (wt), about 0.06% (wt), about 0.07% (wt), about 0.08% (wt),about 0.09% (wt), about 0.1% (wt), about 0.2% (wt), about 0.3% (wt),about 0.4% (wt), about 0.5% (wt), about 0.6% (wt), about 0.7% (wt),about 0.8% (wt), about 0.9% (wt), about 1% (wt), about 2% (wt), about 3%(wt), about 4% (wt), about 5% (wt), about 6% (wt), about 7% (wt), about8% (wt), about 9% (wt), about 10% (wt), about 11% (wt), about 12% (wt),about 13% (wt), about 14% (wt), about 15% (wt), about 16% (wt), about17% (wt), about 18% (wt), about 19% (wt), about 20% (wt), about 21%(wt), about 22% (wt), about 23% (wt), about 24% (wt), about 25% (wt).about 26% (wt), about 27% (wt), about 28% (wt), about 29% (wt), about),about 30% (wt), about 31% (wt), about 33% (wt), about 33% (wt), about34% (wt), about 35% (wt), about 36% (wt), about 37% (wt), about 38%(wt), about 39% (wt), about 40% (wt), about 41% (wt), about 42% (wt),about 43% (wt), about 44% (wt), about 45% (wt), about 46% (wt), about47% (wt), about 48% (wt), about 49% (wt), about 50% (wt), about 51%(wt), about 52% (wt), about 53% (wt), about 54% (wt), about 55% (wt),about 56% (wt), about 57% (wt), about 58% (wt), about 59% (wt), about60% (wt), about 61% (wt), about 62% (wt), about 63% (wt), about 64%(wt), about 66% (wt), about 66% (wt), about 67% (wt), about 68% (wt),about 69% (wt), about 70% (wt), about 71% (wt), about 72% (wt), about73% (wt), about 74% (wt), about 75% (wt), about 76% (wt), about 77%(wt), about 78% (wt), about 79% (wt), about 80% (wt), about 81% (wt),about 82% (wt), about 83% (wt), about 84% (wt), about 85% (wt), about86% (wt), about 87% (wt), about 88% (wt), about 89% (wt), about or 90%(wt). In some embodiments, the adiponectin peptidomimetic compound in anamount ranging from about 0.0001%) (wt) to about 90% (wt), e.g., about0.0001% (wt) to about 90% (wt), about 0.001% (wt) to about 90% (wt),about 0.005% (wt) to about 90% (wt), about 0.01% (wt) to about 90% (wt),about 0.1% (wt) to about 90% (wt), about 1% (wt) to about 90% (wt),about 10% (wt) to about 90% (wt), about 20% (wt) to about 90% (wt),about 30% (wt) to about 90% (wt), about 40% (wt) to about 90% (wt),about 50% (wt) to about 90% (wt), about 60% (wt) to about 90% (wt),about 70% (wt) to about 90% (wt), about 80% (wt) to about 90% (wt),about 10% (wt) to about 50% (wt), about 10% (wt) to about 40% (wt),about 10% (wt) to about 30% (wt), about 10% (wt) to about 20% (wt),about 0.0001% (wt) to about 10% (wt), 0.0001% (wt) to about 1% (wt),0.0001% (wt) to about 0.1% (wt), 0.0001% (wt) to about 0.01% (wt),0.0001% (wt) to about 0.001% (wt), about 0.001% (wt) to about 10% (wt),about 0.001% (wt) to about 1% (wt), about 0.001% (wt) to about 0.1%(wt), 0.01% (wt) to about 0.1% (wt), 0.01%) (wt) to about 1% (wt), andthe like. When administered, the compositions can be given once daily orwith multiple daily doses such as twice per day, three times per day andfour times per day. In some embodiments, the compositions areadministered once a day, every other day, or less frequently. Thecompositions can be administered when the subject has one or moresymptoms of dry eye or an ocular disease. In some instances, thecompositions are given in a dose of one to five drops or more, forexample, one drop, two drops, three drops, four drops, five drops ormore.

When the pharmaceutical composition is used as an ocular ointment, it isdesirable that the ocular ointment contain the adiponectinpeptidomimetic compound in an amount of about 0.0001% (wt) to 90% (wt),e.g., about 0.0001% (wt), about 0.0005% (wt), about 0.001% (wt), about0.005% (wt), about 0.01% (wt), about 0.02% (wt), about 0.03% (wt), about0.04% (wt), about 0.05% (wt), about 0.06% (wt), about 0.07% (wt), about0.08% (wt), about 0.09% (wt), about 0.1% (wt), about 0.2% (wt), about0.3% (wt), about 0.4% (wt), about 0.5% (wt), about 0.6% (wt), about 0.7%(wt), about 0.8% (wt), about 0.9% (wt), about 1% (wt), about 2% (wt),about (wt), about 4% (wt), about 5% (wt), about 6% (wt), about 7% (wt),about 8% (wt), about 9% (wt), about 10% (wt), about 11% (wt), about 12%(wt), about 13% (wt), about 14% (wt), about 15% (wt), about 16% (wt),about 17% (wt), about 18% (wt), about 19% (wt), about 20% (wt), about21% (wt), about 22% (wt), about 23% (wt), about 24% (wt), about 25%(wt), about 26% (wt), about 27% (wt), about 28% (wt), about 29% (wt),about), about 30% (wt), about 31% (wt), about 33% (wt), about 33% (wt),about 34% (wt), about 35% (wt), about 36% (wt), about 37% (wt), about38% (wt), about 39% (wt), about 40% (wt), about 41% (wt), about 42%(wt), about 43% (wt), about 44% (wt), about 45% (wt), about 46% (wt),about 47% (wt), about 48% (wt), about 49% (wt), about 50% (wt), about51% (wt), about 52% (wt), about 53% (wt), about 54% (wt), about 55%(wt), about 56% (wt), about 57% (wt), about 58% (wt), about 59% (wt),about 60% (wt), about 61% (wt), about 62% (wt), about 63% (wt), about64% (wt), about 66% (wt), about 66% (wt), about 67% (wt), about 68%(wt), about 69% (wt), about 70% (wt), about 71% (wt), about 72% (wt),about 73% (wt), about 74% (wt), about 75% (wt), about 76% (wt), about77% (wt), about 78% (wt), about 79% (wt), about 80% (wt), about 81%(wt), about 82% (wt), about 83% (wt), about 84% (wt), about 85% (wt),about 86% (wt), about 87% (wt), about 88% (wt), about 89% (wt), about or90% (wt). In some embodiments, the adiponectin peptidomimetic compoundin an amount ranging from 0.0001% (wt) to about 90% (wt), e.g., about0.0001% (wt) to about 90% (wt), about 0.001% (wt) to about 90% (wt),about 0.005% (wt) to about 90% (wt), about 0.01% (wt) to about 90% (wt),about 0. 1% (wt) to about 90% (wt), about 1% (wt) to about 90% (wt),about 10% (wt) to about 90% (wt), about 20% (wt) to about 90% (wt),about 30% (wt) to about 90% (wt), about 40% (wt) to about 90% (wt),about 50% (wt) to about 90% (wt), about 60% (wt) to about 90% (wt),about 70% (wt) to about 90% (wt), about 80% (wt) to about 90% (wt),about 10% (wt) to about 50% (wt), about 10% (wt) to about 40% (wt),about 10% (wt) to about 30% (wt), about 10% (wt) to about 20% (wt),about 0.0001% (wt) to about 10% (wt), 0.0001% (wt) to about 1% (wt),0.0001% (wt) to about 0.1% (wt), 0.0001% (wt) to about 0.0% (wt),0.0001% (wt) to about 0.001% (wt), about 0.001% (wt) to about 10% (wt),about 0.001% (wt) to about 1% (wt), about 0.001% (wt) to about 0.1%(wt), 0.01% (wt) to about 0.1%) (wt), 0.01% (wt) to about 1% (wt), andthe like. When administered, the compositions may be given once daily orwith multiple daily doses such as twice per day, three times per dayfour times per day, 5 times per day or more. In some embodiments, thecompositions are administered less frequently then once daily. Forinstance, the compositions can be administered every week, every 2weeks, every 3 weeks, every 4 weeks, every 6 weeks, every 7 weeks, every8 weeks, or less frequently. In some embodiments, the compositions areadministered according to the severity of the symptoms experienced bythe subject.

For adiponectin peptidomimetics administered topically, e.g., as eyedrops or ointments, or for intraorbital or perio-ocular injection,exemplary dosages are in the range from about 0.001 to about 100 mg,e.g., in the range from about 0.1 to about 10 mg, for instance, appliedonce a day, twice a day, or more frequently. For ADP 355 administeredtopically, e.g., as eye drops or ointments, or for intraorbital orperio-ocular injection, exemplary dosages can be from about 0.01 mg/mlto about 200 mg/ml.

Having indicated that there is variability in terms of dosing, it isbelieved that those skilled in the art can determine appropriate dosingby administering relatively small amounts and monitoring the patient fortherapeutic effect. If necessary, incremental increases in the dose canbe made until the desired results are obtained. Generally, treatment isinitiated with smaller dosages which may be less than the optimum doseof the therapeutic agent. Thereafter, the dosage is increased by smallincrements until the optimum effect under circumstances is reached. Thetotal daily dosage can be divided and administered in portions duringthe day if desired.

The pharmaceutical preparation can be packaged or prepared in unitdosage form. In such form, the preparation is subdivided into unit dosescontaining appropriate quantities of the active component, e.g.,according to the dose of the therapeutic agent. The unit dosage form canbe a packaged preparation, the package containing discrete quantities ofpreparation. The composition can, if desired, also contain othercompatible therapeutic agents.

The invention provides methods of treating and/or ameliorating dry eyeor an ocular disease associated with inflammation in a subject in needthereof. The course of treatment is best determined on an individualbasis depending on the particular characteristics of the subject. Thetreatment can be administered to the subject on a daily, twice daily,thrice daily, every other day, bi-weekly, weekly, monthly or anyapplicable basis that is therapeutically effective. The treatment can beadministered alone or in combination with at least one other therapeuticagent, e.g., targeting the same ocular disorder or a related symptom.The additional agent can be administered simultaneously with theadiponectin peptidomimetic compound, at a different time, or on anentirely different therapeutic schedule (e.g., the adiponectinpeptidomimetic compound can be administered daily, while the additionalagent is weekly).

F. Co-administration with a Therapeutic Agent

In some embodiments, the methods provided herein includecoadministration of the adiponectin peptidomimetic compound with one ormore additional therapeutic agents. The term “coadministration” refersto administration of a first amount of an adiponectin peptidomimeticcompound or a pharmaceutically acceptable salt thereof and a secondamount of at least one other therapeutic agent, e.g., anothertherapeutic agent for treating an ocular disease, or a therapeutic agentto address associated symptoms, e.g., inflammation. In some instances,the adiponectin peptidomimetic compound and the other therapeutic agentare administered simultaneously or essentially simultaneously. Theadiponectin peptidomimetic compound and the other therapeutic agent maybe in a single pharmaceutical composition, or in multiple pharmaceuticalcompositions. In other instances, the adiponectin peptidomimeticcompound and the other therapeutic agent are administered sequentially.In a sequential dosing, the adiponectin peptidomimetic compound and theother therapeutic agent are administered sufficiently close in time tohave the desired therapeutic effect. For example, the period of timebetween each administration which can result in the desired therapeuticeffect, can range from minutes to hours and can be determined takinginto account the properties of each compound such as potency,solubility, bioavailability, plasma half-life and kinetic profile. Thecombined administration includes coadministration, using separateformulations or a single pharmaceutical formulation, and consecutiveadministration in either order, wherein preferably there is a timeperiod while both (or all) active agents simultaneously exert theirbiological activities.

In some embodiments, the one or more additional therapeutic agentsinclude, punctual plugs, topical steroids topical tetracyclines, topicalnonsteroidal anti-inflammatory drugs (NSAIDS, such as topical diclofenacand topical ketorolac), IL-1 antagonists, other inflammatory pathwayantagonists or inhibitors, angiostatic peptides, angiostatic steroids,modulators/inhibitors of VEGF or FGF, glucocorticosteroids, leukotrienemodulators, anti-histamines, cytokine modulators/inhibitors, growthfactor modulators/inhibitors, T-cell inhibitors, oral or topicalpilocarpine, vitamin A, tretinoin (e.g, all trans-retinoic acid),doxycycline, cyclosporine A (e.g., RESTASIS® (Allergan), azithromycin,mucin stimulants (e.g., Diquafasol (Inspire Pharmaceuticals) 15-(S)-HETE(Alcon), rebamipide (Otsuka) and ecabet (ISTA)), hormonal agents andlacrimal gland stimulants (e.g., androgen tears (Allergan)) and a tearsubstitute (e.g., artificial tears). In some cases, the compositionsdescribed herein are administered in combination with progesterone,synthetic progestogens, medroxyprogesterone acetate, norethindrone,norethindrone acetate, megestrol acetate, 17-a-hydroxyprogesteronecaproate, norgestrel, and derivatives thereof. Additional therapeuticagents include lifitegrast, EBI-005 (Eleven Biotherapeutics), anakinra(Amgen), MIM-D3 (Mimitogen Pharmaceuticals), rebamipide (OtsukaPharmaceuticals), tofacitinib (Pfizer), dexamethasone phosphate (EyeGatePharmaceuticals), RGN-259 (RegeneRx), KPI-121 (loteprednol etabonate;Kala Pharmaceuticals), bromfenac (ISTA Pharmaceuticals), diquafosoltetrasodium (Merck and Co., Inc.), hydroxychloroquine (Sanofi-Aventis),rebamipide (Acucela Inc.), CF101 (Can-Fite BioPharma), lifitegrast(Shire), EBI-005 (Eleven Biotherapeutics), cyclosporine (haporine-S; DHBio Co., Ltd.), rimexolone (Alcon Research), ecabet sodium (Bausch &Lomb Incorporated), rituximab (DEC Pharmaceuticals), tocilizumab(Hoffman-La Roche Ltd.), skQl (Mitotech, SA), cis-UCA (Herantis PharmaPLC), LME636 (Alcon Research), AGN-223575 (Allergan), ISV-101 (InSiteVision), OTX-DP (Ocular Therapeutix, Inc.), rivoglitazone (SantenPharmaceutical Co.), mapracorat (Bausch & Lomb Incorporated), resolvin(Resolvyx), tasocitinib/tofacitinib (Pfizer), RU-101 (R-Tech Ueno,Ltd.), DNase (Genentech, Inc.), voclosporin (Lux Biosciences), P-321(Pari on Sciences), ACCS (Stemnion, Inc.), AGN-23241 1 (Allergan), andthose described in, e.g. Ridder and Karsolia, Clinical Optometry, 2015,2015(7):91-102.

Lifitegrast is a small-molecule that inhibits the integrin, lymphocytefunction-associated antigen 1 (LFA-1), a cell surface protein found onleukocytes. Lifitegrast can bind to LFA-1 which blocks LFA-1 frominteracting with its cognate ligand, intercellular adhesion molecule 1(ICAM-1). This mechanism down-regulates inflammation mediated by Tlymphocytes.

U.S. Pat. No. 8,815,795 pertains to adiponectin, a 247 amino acidsequence suggested for the prevention or treatiment of an eye disease.It is proposed that adiponectin can decrease inflammatory cytokines andhas efficacy for prevention or therapeutic benefit for diseases of theeye such as dry eye (syndrome), eye complications from the use ofcontact lenses, alleviating ocular surface irregularities, decreasinginflammatory cytokines, and increasing conjucntival goblet cell densityas well as providing a composition for eye cleaners or lubricants forcontact lenses wearer.

The combination of an adiponectin peptidomimetic compound with anothertherapeutic agent can result in an enhanced efficacy in the treatment ofocular diseases. Administration of the active agents individually,consecutively, simultaneously, in combination or on an entirelydifferent schedule as compared to the administration for either activeagent individually can result in greater anti-inflammatory activity andimproved clinical efficacy. Clinical studies in animal models indicatedboth improved efficacy and greater anti-inflammatory activity (data notshown). A synergistic effect can also be observed. The synergy can allowfor reduced dosages of the active agents when administered eitherindividually, consecutively, simultaneously, in combination or on anentirely different schedule as compared to the dosages for either activeagent individually. The reduced dosage can help reduce any side effectsthat may appear. Accordingly, in combination therapy, the effectiveamount of the additional (second) therapeutic agent and the effectiveamount of the adiponectin peptidomimetic compound are together effectiveto reduce the symptoms/effects of an ocular disease.

One of skill in medicine can best determine the appropriate dose of theadditional therapeutic agent by considering the state of the patient,the recommended dose, the severity of disease, and the efficacy ofadministering the combination of the adiponectin peptidomimetic compoundwith the therapeutic agent. Synergistic effects resulting from theadministering the combination of the adiponectin peptidomimetic compoundwith the therapeutic agent can also result.

G. Methods of Determining Therapeutic Efficacy

A variety of methods can be performed evaluate a subject's treatmentresponse to the compositions provided herein. In some instances, anassay, test or measurement can be made to determine whether methodsdescribed herein have alleviated at least one symptom or clinical signof dry eye or an ocular disease associated with inflammation. Detaileddescriptions of methods for measuring or evaluating symptoms or clinicalsigns of dry eye or an ocular disease provided herein are found in, forexample, Pult et al., Eye (Lund), 2011, 25(4): 502-510, Bhatnagar etal., Int J Opthalmol, 2015, 8(1): 174-81, Messmer, Dtsch Arztebl Int,2015, 112(5): 71-82.

Changes in tear secretion can be assessed by the Schirmer's test, phenolred thread tear rest (PRTT) and other methods of determining the rateand quantity of tear production. Changes in tear clearance can beassessed by fluorescein clearance test and fluorophotometry. Ocularsurface damage and corneal epithelial defects can be evaluated vital dyestaining, e.g., fluorescein, rose bengal, and lissamine green staining.Cytology of the ocular surface can be analyzed by impression cytology,brush cytology, flow cytometry, and confocal microscopy. Tear filmstability can be looked at by analyzing tear break-up time, using theTear film Stability Analysis System (TSAS). wavefront aberrometry, laserscanning microscopy, functional visual acuity, and tear filminterferometry. Tear volume change can be assessed by tear meniscusmeasurement. Lipid layer changes to tear film can be assessed by tearfilm interferometry, meibometry, and meibography. Tear evaporationassessment can be made by evaporimeter, closed chamber, and ventilatedchamber. Improvements in tear film chemical properties can be assessedby tear osmolarity, depression of freezing point, vapor pressureosmometry, and conductivity (Ocusense). Biochemical analysis of tearcomposition may include mucin and lipid analyses. Improvement in theocular surface can be visualized by using dyes (such as fluorescein,lisamine green or rose bengal and observing less irregular morphologyand staining of the corneal or conjunctival epithelium, compared tobaseline.

The alleviation of at least one symptom or a clinical sign of an oculardisease, such as dry eye and an ocular disease associated withinflammation, can be determined by comparing the degree of the symptomor clinical sign after treatment to the degree of the same symptom orclinical sample prior to treatment. If the degree of the symptom orclinical sign has decreased after treatment, then an improvement oralleviation can be indicated.

IV. Examples

The following examples are offered to illustrate, but not to limit, theclaimed invention.

Example 1 Adiponectin Ppeptidomimetic Compounds to Treat an Animal Modelof Dry Eye

The purpose of this study is to use the scopolamine model of dry eye tostudy the efficacy of an adiponectin peptidomimetic compound describedherein.

A. Treatment Protocol

Male Sprague-Dawley rats weighing between 300 g and 350 g are obtainedfrom Charles River (Wilmington, Mass.). Animals are housed in animalquarters under constant room temperature (22±1° C.), light conditions(12-h light/12-h dark cycle), and humidity (40-60%). Animals areanaesthetized before the surgical experiment and clinical examinationwith isofluorane.

Dry eye is induced using scopolamine (Sigma-Aldrich, St. Louis, Mo.),which is continuously and systemically delivered to the animals via anosmotic pump (2ML4 Alzet®; CedarLane, Burlington, Ontario) filled withscopolamine and implanted subcutaneously in the mid dorsal area betweenthe scapulae. The wound is closed with 2-3 wound clips. After thesurgery and again the next day, the animals are subcutaneously injectedwith Carprofen (0.5 mg/100 g) a non-steroidal anti-inflammatory drug andpotent, long-acting analgesic in rodents. Animals are anaesthetizedbefore the surgical pump implantation and before all clinical endpointtesting in an Isofluorane 99.9% USP (Abraxis Bioscience, Richmond Hill,Ontario) chamber. Scopolamine is delivered at 12.5 mg/day and, fortechnical reasons, the data is evaluated at day 14.

The sterile solution of 0.175 g/mL of scopolamine hydrobromide(Sigma-Aldrich, St. Louis Mo.) is prepared in saline (0.9%) and filteredthrough a 0.22 um syringe-end filter (Millex-GC, Millipore Corp.,Bedford, Mass.). A 2ML4 Alzet® pumps is filled with 2 mL of 0.175 g/mLscopolamine solution according to the manufacturer's instructions.

B. Clinical Endpoints for Dry Eye and Results

Corneal Staining: clinical signs of corneal dryness are evaluated byfluorescein impregnation of the cornea. A drop of a 1% fluoresceinsodium (Sigma-Aldrich, St. Louis, Mo.) solution made up in sterilesaline is instilled in the conjunctival sac of the anaesthetized animal.The cornea is thereafter observed under blue light using a Portable SlitLamp ophthalmoscope with blue cobalt filter (Reichert OphthalmicInstruments, Depew, N.Y.) three minutes after fluorescein instillation.For each animal, the punctate fluorescent-positive area of the ocularsurface is recorded in a blinded fashion. The score of this test isgraded from 0 to 4, where 0=no staining, 1=<25% surface staining,2=25-50% surface staining, 3=50-75%) surface staining and 4=>75%>surfacestaining.

Schirmer's test: Tear production is measured with Zone-Quickstandardized phenol-red threads (FCI Ophthalmics, Marshfield Hills,Mass.). Animals are lightly sedated with Isoflurane. The threads areinserted in the lateral lower canthus and left in place for thirtyseconds. The length of the stained moistened portion of the thread ismeasured in millimeters, using the scale provided with the threads to anaccuracy of 1 mm.

The mean and standard deviation (SD) is used to characterize the datafor each study group. A one-way analysis of variance (ANOVA) isperformed for body weight and the ophthalmic signs for treatment groupsat every observation using Graph Pad Prism 4.0C (Graph Pad SoftwareInc., La Jolla, Calif). When stratified by examination day, when thetreatment group is statistically significant (p<0.05, two-tail), pairwise comparisons are performed. For comparison to the untreated control(Group 1 or A), adjustment with Dunnett's test is used. No correctionsare made for multiple comparisons. P values between groups is calculatedand the difference between each of a pair of means (reported P valuesas >0.05, <0.05, <0.01 or 0.001) is reported.

Example 2 Adiponectin Peptidomimetic Compounds to Treat a Mouse Model ofDry Eye Using Scopolamine Combined with a Desiccating Environment

Six- to eight-week-old female C57BL/6 mice are used in theseexperiments. Experimental dry eye (EDE) is induced by subcutaneousinjection of 0.5 mg/0.2 mL scopolamine hydrobromide (Sigma-Aldrich, St.Louis, Mo.) four times a day (8 AM, 11 AM, 2 PM, and 5 PM) with exposureto an air draft and 30% ambient humidity, as previously described.During these experiments, the animals' behavior, food, and water intakeare not restricted. The mice are randomly assigned to six groupsaccording to topical treatment administered as follows: (1) untreated(UT) control mice that are not exposed to desiccating stress or treatedtopically; (2) EDE control mice that receive no eye drops; (3) EDE micetreated with balanced salt solution (BSS; Alcon, Fort Worth, Tex.), (4)EDE mice treated with 0.001%) adiponectin peptidomimetic; (5) EDE micetreated with 0.01% adiponectin peptidomimetic; and (6) EDE mice treatedwith 0.1% adiponectin peptidomimetic. All treatment groups receive 2microliters eye drops four times a day. Tear volume and cornealsmoothness are measured at 5 and 10 days after treatment. Ten days aftertreatment, the mice are euthanized, and multiplex immunobead assay,histology, immunohistochemistry, and flow cytometry were performed. Eachgroup consists of five animals, and the experiments are performed onfour independent sets of mice.

Tear volume is measured using phenol red-impregnated cotton threads(Zone-Quick; Oasis, Glendora, Calif.), as previously described. Thethreads are placed in the lateral canthus for 20 seconds. The distancesof threads wet by tears are measured using the SMZ 1500 microscope(Nikon, Tokyo, Japan). A standard curve is derived to convert distanceinto volume.

Severity of corneal surface irregularity is graded via measurement ofthe distortion of a white ring from the fiberoptic ring illuminator ofthe stereoscopic zoom microscope (SMZ 1500; Nikon) by two maskedobservers. The corneal irregularity severity score is calculated using a6-point scale (0-5) based on the number of distorted quarters in thereflected ring, as follows: 0, no distortion; 1, distortion in onequarter of the ring; 2, distortion in two quarters; 3, distortion inthree quarters; 4, distortion in all four quadrants; 5, severedistortion, in which no ring could be recognized.

Flow cytometry is performed for quantitation of CD4⁺CXCR3⁺ T cells fromthe conjunctiva and lacrimal gland. The tissues are teased and shaken at37° C. for 60 minutes with 0.5 mg/mL collagenase type D. After grindingwith a syringe plunder and passage through a cell strainer, cells areobtained, centrifuged, and resuspended in PBS with 1% bovine serumalbumin. After washing, the samples are incubated withfluorescein-conjugated anti-CD4 antibody (BD Biosciences, San Jose,Calif.), phycoerythrin-conjugated anti-CXCR3 antibody (BD Biosciences),and isotype control antibody at 37° C. for 30 minutes. The number ofCD4⁺CXCR3⁺ T cells is counted by a FACSCalibur cytometer with CellQuestsoftware (BD Biosciences)

Statistical differences in the tear volume and corneal irregularityscore results are evaluated by one-way ANOVA, with post hoc analysis.Kruskal-Wallis and Mann-Whitney test are used to compare the cytokinelevel, goblet cell density, and flow cytometry between groups. Ap-value<0.05 is considered statistically significant.

Administration of adiponectin peptidomimetics described herein canincrease tear volume, decrease severity of corneal surface irregularity,and/or decrease the number of inflammatory response cells in and aroundthe eye in a mouse model of dry eye.

Example 3 Topical Administration of Adiponectin Peptidomimetics ImprovesTear Production and Corneal Surface Irregularities Caused by Dry Eye

This example illustrates the use of adiponectin peptidomimetics such asADP355 and ADP399 to treat dry eye in a subject in need thereof. ADP355has the sequence: DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-NH₂ (SEQ IDNO:6). ADP399 is a linear branched dimer of ADP355 and has the sequence:(DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-His-Pro)₂-Dab-NH₂ (SEQ IDNO:7). Nva refers to norvaline and Dab refers to 2,3-diamino butyricaicd. Detailed descriptions of ADP355 and ADP399 are found, for example,in U.S. Pat. No. 9,073,965 and Otvos et al., Frontiers in Chemistry,2014, 2(93): 1-15, doi: 10.3386/fchem.2014.00093, the disclosures arehereby incorporated by reference in their entirety for all purposes.

Experimental dry eye (EDE) was induced in 8-week old female C57BL/6 miceby subcutaneous injection of 0.5 mg/0.2 mL scopolamine hydrobromide(Sigma-Aldrich, St. Louis, Mo.) three times a day (9 AM, 1:30 PM and 6PM). A dose of 0.3 mL scopolamine hydrobromide was administered at eachinjection. The mice were also exposed to an air draft and 30% ambienthumidity. 6 experimental groups used in this study included: (1)untreated control mice (UT; no dry eye treatment and no topicaltreatment); (2) untreated EDE control mice (EDE; received no topicaladministration); (3) EDE control mice received a balanced salt solution(BSS); (4) EDE mice received 0.01% single chain adiponectinpeptidomimetic (ADP355) in BSS; (5) EDE mice receiving 0.01% peptidedimer adiponectin peptidomimetic (ADP399) in BSS; and (6) EDE micereceiving 0.01% recombinant globular adiponectin (gAdipo) in BSS. Eachgroup included 5 mice. EDE mice treated with BSS received topicalbilateral administration of 2 μï/eye, 3 times a day. EDE mice treatedwith ADP355 received topical bilateral administration of 2 μï/eye, 3times a day. EDE mice treated with ADP-399 received topical bilateraladministration of 2 μï/eye, 3 times a day. The gAdipo treated miceserved as a positive treatment control. Mice were evaluated at baseline,day 5 and day 10 after the treatment initiation. In some experiments,ADP355, ADP-399 and gAdipo were resuspended in 0.01% BSA.

Tear volume was measured using a phenol red thread tear test. Briefly, acotton thread treated with phenol red at its tip (Zone-Quick; Oasis,Glendora, Calif.) was held with jeweler's forceps and placed in the tearmeniscus of the lateral canthus for 20 sees to contact tear fluid. Usinga microscope, the length of the red portion (wet portion) of the threadwas measured in millimeters. A standard curve was derived to convert thedistance measurement into tear volume.

FIG. 1 shows that tear production, as measured by tear volume, washigher in adiponectin peptidomimetic treated EDE mice compared tountreated or BSS treated EDE mice (p-value<0.05 for ADP355 vs. EDE,p-value<0.05 for ADP399 vs. EDE). A statistically significant differenceof tear volume was observed between the treated and untreated EDE mice.There was no significant difference in tear volume among those treatedwith ADP355, ADP399 and globular adiponectin. Tear volumes in all groupsat day 10 after treatment initiation were similar to those at day 5.

The severity of corneal epithelial damage was evaluated by cornealfluorescein staining. Briefly, 1% fluorescein dye (1 μï) was instilledinto the eye and then the eye was washed with saline. 10 minutes afterapplication of the dye, the eye was photographed with a slip lampmicroscope using a cobalt blue light. The stained area was graded usinga scoring system by two blinded observers. Five different regions of thecornea (central, superior, inferior, nasal and temporal regions) wereassessed according to a 0-4 score for each region: score 0 represents noor absent staining; score 1 represents punctate staining of less than 30spots; score 2 represents punctate staining of greater than 30 spots butnot diffuse; score 3 represents severe diffuse staining but not orabsent positive plaque; and score 4 represents severe diffuse stainingwith positive fluorescent plaque. The average score from the fiveregions was recorded. FIG. 2 shows that EDE mice treated with anadiponectin peptidomimetic such as ADP355 and ADP-399 exhibited lesscorneal staining (less corneal irregularity) compared to untreated EDEmice (p-value<0.05 for ADP355 vs. EDE, p-value<0.05 for ADP-399 vs.EDE). A statistically significant improvement of the corneal fluorescentstaining score was observed in the adiponectin peptidomimetic treatedgroup compared to the untreated group. In addition, the adiponectinpeptidomimetic-treated mice (ADP355 and ADP-399) had corneal surfacesthat were similar to those of untreated normal mice (UT) and positivecontrol globular adiponectin-treated mice (gAdipo). The corneal surfacescores in all groups at day 10 were similar to those at day 5 aftertreatment initiation.

This study shows that topical administration of an adiponectinpeptidomimetic reduced or eliminated a clinical sign of dry eye such astear production and ocular surface irregularities. Administration of eyedrops containing adiponectin peptidomimetics including single chainadiponectin peptidomimetics (e.g., ADP355) and peptide dimer adiponectinpeptidomimetics (e.g., ADP399) had a beneficial effect on tearproduction and ocular surface of EDE. As such, topical application ofadiponectin peptidomimetics to the eye can be useful for the treatmentof dry eye in a subject in need thereof.

Example 4 Topical Administration of Adiponectin Peptidomimetics inCombination with 5% Lifitegrast Improves Tear Film Stability and CornealSurface Irregularities Caused by Dry Eye

This example illustrates the use of adiponectin peptidomimetics such asADP355 in combination with 5% Lifitegrast to treat dry eye in a subjectin need thereof. ADP355 has the sequence:DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-NH₂ (SEQ ID NO:6).Lifitegrast is a prescription eye drop sold under the trade name Xiidra(Shire US Inc., Lexington, Mass.) as a 5% ophthalmic solution fortopical ophthalmic use. A detailed description of ADP355 is found, forexample, in U.S. Pat. No. 9,073,965 (ADP355), the disclosure is herebyincorporated by reference in its entirety for all purposes.

Experimental dry eye (EDE) was induced in 8-week old female C57BL/6 miceby subcutaneous injection of 0.5 mg/0.2 mL scopolamine hydrobromide(Sigma-Aldrich, St. Louis, Mo.) three times a day (9 AM 0.3 mL, 1:30 PM0.3 mL, and 6:00 PM 0.3 mL). The mice were also exposed to an air draftand 30% ambient humidity. Five experimental groups used in this studyincluded: (1) untreated control mice (UT; no dry eye treatment and notopical treatment); (2) EDE control mice received a balanced saltsolution (BSS); (3) EDE mice received ADP 355 0.1% (single chainadiponectin peptidomimetic (ADP 355) in BSS); (4) EDE mice receivedLifitegrast 5%; and (5) EDE mice received 0.1% peptide adiponectinpeptidomimetic (ADP 355) in BSS+Lifitegrast 5%. Each group included 5mice. EDE mice treated with BSS, or 0.1% Adiponectin 355 alone or incombination with Lifitegrast 5%, received topical bilateraladministration of BSS, or 0.1% Adiponectin 355, 2 μï/eye, 3 times a day.EDE mice treated with Lifitegrast 5%, alone or in combination with 0.1%Adiponectini355, received topical bilateral administration ofLifitegrast 5%, 2 μ{umlaut over (l)}/eye, 2 times a day. Mice wereevaluated at baseline, day 5 and day 10 after the treatment initiation.

Experiment 1—Tear Film Break-up (BUT): This experiment evaluated theanti-inflammatory activity and efficacy of the ADP355 0.1% alone or incombination with Lifitegrast 5% in ameliorating Desiccation-induced DryEye (DED). Eyes were evaluated for time for dry spots to appear on thecorneal surface of the eye after blinking. This assay provides a methodto evaluate the tear film stabiliy as well as determining if evaoprativedry eye is present. The longer the time before tear film break-upindicates a more stable tear film. Subjects with normal tear quantitybut an unstable tear film can explain dry eye symptoms. Tear instabilitycan indicate an imbalance in tear composition causing tears to evaporatetoo fast or improper adhesion to the eye surface. Alternatively,insufficient lipid secretion by the melbomian glands due to melbomiamgland dysfunction can also cause tear film instability. Evaluations weredone at five and 10 day enpoints with around >3-5 seconds a normal time.<2.2-3.0 seconds marginal and <2.2 seconds low and a high likelihood ofdry eye symptoms and no discernable improvement in efficacy.

FIGS. 3A and 3B illustrate that tear production, as measured by tearfilm break-up time (BUT). BUT (stability), was longer in adiponectinpeptidomimetic ADP 355+Lifitegrast 5% treated EDE mice compared to BSStreated EDE mice (p-value<0.05 for Groups 3-6). Group 5 (those treatedwith ADP355+Lifitegrast 5%) had a noticeable improvement in tear filmtimes at both endpoints (around 4.8 seconds at D5 and 4.2 seconds atD10) compared to Groups 3 and 4 treated with either 0.1% Adiponectin355, or with Lifitegrast 5% alone, respectively. Clearly there appearedto be an additive effect by treating with both ADP355 0.1%+Lifitegrast5%.

Experiment 2—Corneal staining: The purpose of the corneal staining assaywas to evaluate damage to the cornea epithelia in the murine EDE subjecteither as a consequence of inducing dry eye or due to treatment with0.1% ADP 355 or with Lifitegrast 5% alone or with the combination of0.1% ADP 355+Lifitegrast 5%. Fluorescein staining detects disruptions,changes in the contu e and detectable injury to the epithelium surfacecells of the cornea. The lower the staining grade the less severe thecorneal damage. Corneal staining was assessed in four quadrants using a4-point scale with 16 being the maximum score.

Briefly, fluorescein dye (1 μï) was instilled into the eye and then theeye was washed with saline. 10 minutes after application of the dye, theeye was photographed with a slip lamp microscope using a cobalt bluelight. The stained area was graded using a scoring system by two blindedobservers. Five different regions of the cornea (central, superior,inferior, nasal and temporal regions) were assessed according to a 0-4score for each region: score 0 represents no or absent staining; score 1represents slightly punctate staining of less than 30 spots; score 2represents punctate staining of greater than 30 spots but not diffuse;score 3 represents severe diffuse staining but no or absent positiveplaque; and score 4 represents severe diffuse staining with positivefluorescein plaque. The average score from the five regions wasrecorded. FIGS. 4A and 4B illustrate grading of the staining results atD5 and D10, respectively. Again, as in the tear film break-up study,treated eyes had a lower grade than the untreated eyes (Group 2, BSS).Treatment with either ADP 355 0.1%, or Lifitegrast 5% improved cornealdisruptions resulting from induced dry eye. However, greater improvementwas most noticeable at D5 (FIG. 4A) in EDE eyes treatment with both 0.1%ADP 355+Lifitegrast 5%. At D10 (FIG. 4B), the combination was onlyslightly better than any of single treatments alone and each treatmentstill had a lower grade than the untreated dry eyes.

Experiment 3—Flow Cytometry: The purpose of this test was to quantifythe presence of CD4+ and CCR5+ T cells from the conjunctiva and lacrimalgland as previously described (Yoon K C, Heo H, Kang I S, et al. Cornea(2008) 27: 454-460). High levels of inflammatory cells is indicative ofocular inflammation. The tissues are teased and shaken at 37° C. for 60minutes with 0.5 mg/mL collagenase type D.

After grinding with a syringe plunder and passage through a cellstrainer, cells are obtained, centrifuged, and resuspended in PBS with1% bovine serum albumin. After washing, the samples are incubated withfluorescein-conjugated anti-CD4 antibody (BD Biosciences, San Jose,Calif.), phycoerythrin-conjugated anti-CC5+R antibody (BD Biosciences),and isotype control antibody at 37° C. for 30 minutes. The number ofCD4⁺ and CCR5⁺ T cells is counted by a FACSCalibur cytometer withCellQuest software (BD Biosciences). Results for each treatment groupare presented in FIGS. 5A-5E and 6A-6E. The results indicate that after10 days only 0.1% ADP 355+Lifitegrast 5% restablished Activated T Celllevels within the normal range as the percentage was 20.06% with thecontrol at 28.86% in the conjunctiva (FIGS. 5E and 5A, respectively)12.53% with the control at 11.09% in the lacrimal gland (FIGS. 6E and6A, respectively).

Experiment 4—Cytokine Assessment

The purpose of this experiment is to identify and assess the presence ofcytokines in the conjunctive and lacrimal gland of mice with and withoutEDE.

A multiplex immunobead assay (Luminex 200; Luminex Corp., Austin, Tex.)is used to measure the concentrations of IFN-γ, IL-1B, MIP1B, TNF-α,IL-6, IFN-c, and monokine induced by interferon-c (MIG) in theconjunctiva and lacrimal gland. The tissues are collected and pooled inlysis buffer containing protease inhibitors for 30 minutes. The cellextracts are centrifuged at 14,000 g for 15 minutes at 48° C., and thesupernatants were stored at −70° C. before use. The supernatants areadded to wells containing the appropriate cytokine bead mixture thatinclude mouse monoclonal antibodies specific for IFN-γ, IL-1B, MIP1B,TNF-α, IL-6, IFN-c, and MIG for 60 minutes. After three washes withassay buffer, the biotinylated secondary cytokine antibody mixture isapplied for 30 minutes in the dark at room temperature. The reactionsare detected after addition of streptavidin-phycoerythrin with ananalysis system (xPONENT, Austin, Tex.). The concentrations of thesefactors in tissue are calculated from standard curves of knownconcentrations of recombinant mouse cytokines.

Eye and adnexa are surgically excised, fixed in 4% paraformaldehyde, andembedded in paraffin. Six-micrometer sections are stained with periodicacid-Schiff (PAS) reagent. Sections are examined and photographed with amicroscope (BX53; Olympus, Tokyo, Japan) equipped with a digital camera(F2; Foculus, Finning, Germany). Goblet cell density in the superior andinferior conjunctiva are measured in three sections from each eye usingimage analysis software (Media Cybernetics, Silver Spring, Md.) andexpressed as the number of goblet cells per fixed area.Immunohistochemistry is performed to detect the expression ofadiponectin receptors, AdipoRI and AdipoR2, in the conjunctiva of normaleyes and TNF-a in the conjunctiva and lacrimal gland of experimental dryeyes. Hydrogen peroxide (H₂0₂, 0.3%) in phosphate buffered saline (PBS)and 20% serum in PBS are sequentially applied to the sections.Conjunctival sections from UT control mice are incubated with goatanti-adiponectin receptor AdipoRI and AdipoR2 antibodies (VectorLaboratories, Burlingame, Calif.). Conjunctival and lacrimal glandsections from mice with EDE are incubated with goat monoclonalanti-mouse TNF-a antibody (Santa Cruz Biotechnology, Santa Cruz,Calif.). After washing, appropriate secondary antibodies are applied.The samples are incubated with avidin-peroxidase, then incubated with3,30-diaminobenzidine peroxidase substrate and counterstained withMayer's hematoxylin.

FIG. 7 illustrates cytokine levels in conjunctiva and lacrimal glandsfollowing treatment with a BSS, 0.1% ADP 355, Lifitegrast 5% or 0.1% ADP355+administration of Lifitegrast 5%. Readily evident is the decreasedlevel of cytokines resulting from treatment with administration of both0.1% ADP 355+Lifitegrast 5%, providing improved efficacy of the combinedtreatment.

Statistical differences in the tear break-up time, corneal staining andnormal score results were evaluated by one-way ANOVA, with post hocanalysis. Kruskal-Wallis and Mann-Whitney test were used to compare flowcytometry between groups. A P value<0.05 was considered statisticallysignificant.

This study shows that topical administration of an adiponectinpeptidomimetic, 0.1% ADP 355+ administration of Lifitegrast 5% improvedtear film stability and eliminated a clinical sign of dry eye such asocular surface irregularities and decreased the number of inflammatoryresponse cells in and around the eye in a mouse model of dry eye whencompared to topical administration of either 0.1% ADP 355, orLifitegrast 5% alone. As such, topical application of an adiponectinpeptidomimetic(s)+Lifitegrast 5% to the eye appears to have improvedefficacy and can be useful for the treatment of dry eye in a subject inneed thereof.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

What is claimed is:
 1. A method for treating dry eye in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of a composition comprising: anadiponectin peptidomimetic compound, Lifitegrast and a pharmaceuticallyacceptable carrier, to treat dry eye in the subject, wherein theadiponectin peptidomimetic compound is represented by Formula II:Xaa_(i)-Ile-Pro-Xaa₂-Leu-Tyr-Xaa₃-Phe-Ala-Xaa₄-Xaa₅ (SEQ ID NO:1),wherein Xaa_(i) is Asn or a non-natural amino acid, Xaa₂ is Gly or anon-natural amino acid, Xaa₃ is Tyr or a non-natural amino acid, Xaa₄ isTyr or a non-natural amino acid, and Xaa₅ is no amino acid, β-Ala orP-Ala-NH₂, wherein the C-terminal amino acid is optionally amidated, ora pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the composition is administered topically, by intravitrealinjection, by subconjunctival injection, by conjunctival injection, byintramuscular injection, by subcutaneous injection, by intravenousinjection, by intracameral injection, or by implantation into thesubject's eye.
 3. The method of claim 1, wherein the dry eye is selectedfrom the group consisting of hypolacrimation, tear deficiency,xerophthalmia, Sjogren's syndrome dry eye, non-Sjogren's syndrome dryeye, keratoconjuctivitis sicca, aqueous tear- deficiency dry eye (ADDE),evaporative dry eye (EDE), environmental dry eye, Stevens- Johnsonsyndrome, ocular pemphigoid blepharitis marginal, eyelid-closurefailure, sensory nerve paralysis, allergic conjunctivitis-associated dryeye, post- viral conjunctivitis dry eye, postcataract surgery dry eye,VDT operation- associated dry eye, and contact lens wearing- associateddry eye.
 4. The method of claim 1, wherein the composition isadministered to the subject once a day, two times a day, three times aday, four time a day or more often.
 5. The method of claim 1, whereinthe composition is administered to the subject once a day, two times aday, three times a day, four time a day or more often.
 6. The method ofclaim 1, wherein the composition is administered every other day or lessoften.
 7. The method of claim 1, wherein the composition is administeredevery other day or less often.
 8. The method of claim 1, wherein theadiponectin peptidomimetic compound is present in an amount between0.0001% (wt) to 90% (wt) of the composition.
 9. The method of claim 1,wherein the composition is administered in an amount between 0.1% (wt)to 20% (wt) of the composition.
 10. The method of claim 1, wherein thecomposition is in a formulation selected from the group consisting of asolution, suspension, syrup, liquid, gel, hydrogel, emulsion, liposome,aerosol, mist, film, suspension, plug, polymer, implant, contact lens,ocular insert, nanoparticle, microparticle, a sustained releaseformulation, and a formulation suitable for an ocular medical device.11. The method of claim 1, further comprising administering acomposition comprising cyclosporine, artificial tears, a corticosteroid,an anti-inflammatory agent, or any combination thereof.
 12. The methodof claim 1, wherein the adiponectin peptidomimetic compound is selectedfrom the group consisting of D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser (SEQ ID NO:3), D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-DSer-p-Ala (SEQ ID NO:4), D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Serp-Ala-NH2(SEQ ID NO:5), D-Asn-Ile-Pro-Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser-NH2 (SEQ ID NO:6),(D-Asn-Ile-Pro- Nva-Leu-Tyr-D-Ser-Phe-Ala-D-Ser-His-Pro)₂-Dab- NH2(SEQID NO:7), a variant thereof, a derivative thereof, and apharmaceutically acceptable salt thereof.
 13. The method of claim 1,wherein the adiponectin peptidomimetic compound is SEQ ID NO:6.
 14. Themethod of claim 1, wherein the composition further comprises atherapeutic agent which is a cyclosporine ophthalmic emulsion.
 15. Themethod of claim 14, wherein the therapeutic agent comprises 3.5% to 6.5%Lifitegrast or a pharmaceutically acceptable salt thereof.
 16. Themethod of claim 1, wherein the subject presents at least one symptom orclinical sign of dry eye selected from the group consisting of a changein tear secretion, a change in tear clearance, ocular surface damage,corneal epithelial defects, a change in ocular surface cells, a changein tear film stability, a change in tear volume, a change in tear filmcomposition, a change in tear osmolarity, and any combination thereof.